Oral immunization by transgenic plants

ABSTRACT

The invention is directed to transgenic plants expressing colonization and/or virulence antigens specified by genes from pathogenic microorganisms. It is also directed to the use of such transgenic plants for oral immunization of humans and other animals to elicit a secretory immune response which inhibits colonization of or invasion by such pathogenic microorganisms through a mucosal surface of humans or other animals.

This is a continuation of application Ser. No. 07/398,520, filed 29 Aug.1989, which is a continuation-in-part of application Ser. No.07/240,728, filed 6 Sep. 1988 (now abandoned).

BACKGROUND OF THE INVENTION

Advances in recombinant DNA technology coupled with advances in planttransformation and regeneration technology have made it possible tointroduce new genetic material into plant cells, plants or plant tissue,thus introducing new traits, e.g., phenotypes, that enhance the value ofthe plant or plant tissue. The present invention relates to theintroduction i to plants of genes encoding colonization or virulenceantigens or parts thereof of pathogens which colonize on or invadethrough mucosal surfaces of animal species. The present invention alsorelates to production of such colonization or virulence antigen or partsthereof by the plants. The invention further relates to the use of plantmatter containing such colonization or virulence antigen or partsthereof for the oral immunization of humans and other animals to inhibitinfection of the animal or human by the pathogen.

A. General Overview of Infectious Diseases and Immunity

Infectious disease are becoming an increasing problem for both animaland human health. Gillespie J. et al., Infectious Disease of DomesticAnimals, Comstock Press, Ithaca, N.Y. (1981); Mandell, G. L. et al.,Principles and Practices of Infectious Diseases, 2nd Ed., John Wiley andSons, New York (1985). Diseases caused by bacterial pathogens areparticularly troublesome due to the increase in antibiotic-resistantpathogens. Most pathogens enter on or through a mucosal surface, withthe exception of the insect-borne pathogens or those which enter thebody through a wound. The former pathogens include, but are not limitedto, pathogenic species in the bacterial genera Actinomyces, Aeromonas,Bacillus, Bacteroides, Bordetella, Brucella, Campylobacter,Capnocytophaga, Clamydia, Clostridium, Corynebacterium, Eikenella,Erysipelothrix, Escherichia, Fusobacterium, Hemophilus, Klebsiella,Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria,Nocardia, Pasteurella, Proteus, Pseudomonas, Rickettsia, Salmonella,Selenomonas, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio,and Yersinia, pathogenic virul strains from the groups Adenovirus,Coronavirus, Herpesvirus, Orthomyxovirus, Picornovirus, Poxvirus,Reovirus, Retrovirus, Rotavirus, pathogenic fungi from the generarAspergiullus, Blastomyces, Candida, Coccidiodes, Cryptococcus,Histoplasma and Phycomyces, and pathogenic parasites in the generaEimeria, Entamoeba, Giardia, and Trichomonas. It is generallyacknowledged that prevention of infectious diseases would be much morecost-effective that attempts to treat infections once they occur. Thus,increased attention is being addressed to the development of vaccinesfor the effected immunization of humans and other animals. Germanier, .,Bacterial Vaccines, Academic Press, London (1984); Brown, F., Ann. Rev.Microbiol. 38, 221 (1984).

Animal human hosts infected by a pathogen mount an immune response in anattempt to overcome the pathogen. There are three branches of the immunesystem: mucosal, humoral and cellur. Hood, L. E. et al., Immunology, 2ndEd., Benjamin Publishing Co., Menlo Park, Calif. (1984).

Mucosal immunity results from the production of secretory IgA (sIgA)antibodies in secretions that bathe all mucosal surfaces of therespiratory tract gastrointestinal tract and the genitourinary tract andin secretions from all secretory glands. McGhee, J. R. et al., Annals NYAcad. Sci. 409, (1983). These sIgA antibodies act to preventcolonization or invasion through a mucosal surface. The production ofsIgA can be stimulated either by local immunization of the secretorygland or tissue or by presentation of an antigen to either thegut-associated lymphoid tissue (GALT or Peyer's patches) or thebronchial-associated lymphoid tissue (BALT). Cebra, J. J. et al., Coldspring Harbor Symp. Quant. Biol. 41, 210 (1976); Bienenstock, J. M.,Adv. Exp. Med. Biol. 107, 53 (1978); Weisz-Carrington, P. et al., J.Immunol 123, 1705 (1979);; McCaughan, G. et al., Internal Rev. Physiol28, 131 (1983). Membranous microfold cells otherwise known as M Cells,cover the surface of the GALT and BALT and may be associated with othersecretory mucosal surfaces. M cells act to sample antigens from theluminal space adjacent to the mucosal surface and transfer such antigensto antigen-presenting cells (dendritic cells and macrophages), which inturn present the antigen to a T lymphocyte (in the case of T-dependentantigens), which process the antigen for presentation to a committed Bcell. B cells are then stimulated to proliferate, migrate and ultimatelybe transformed into an antibody-secreting plasma cell producing IgAagainst the presented antigen. When the antigen is taken up by M cellsoverlying the GALT and BALT, a generalized mucosal immunity results withsIgA against the antigen being produced by all secretory tissues in thebody. Cebra et al., supra; Bienenstock et al., supra; Weinz-Carringtonet al., supra; McCaughan et al., supra. Oral immunization is therefore amost important route to stimulate a generalized mucosal immune responseand, in addition, leads to local stimulation of a secretory immuneresponse in the oral cavity and in the gastrointestinal tract.

Humoral immunity results from production of IgG and IgM in serum andpotentiates phagocytosis of pathogens, the neutralization of viruses, orcomplement-mediated cytotoxicity of pathogens (Hood et al., supra). Theimmunity to a pathogen can be transmitted from the mother to theoffspring in both birds and mammals by delivery of the secretoryantibody either in the egg or in the colostrum or by placental transferof serum antibody in the case of mammals. McGhee et al., supra, McNabbet al., supra; Mestekcy. J., J. Clin. Immunol., 7, 265 (1987).

Cellular immunity is of two types: One is termed a delayed-typehypersensitivity response which causes T Lymphocytes to stimulatemacrophages to kill bacterial, parasitic, and mycotic pathogens. In theother type, cytotoxic T lymphocytes are directed to kill host cellsinfected with viruses. Hood, et al. supra.

Secretory IgA antibodies directly inhibit the adherence ofmicroorganisms to mucosal epithelial cells and the teeth of the host.Abraham, S. N. et al., Advances in Host Defense Mechanisms, Raven Press,N.Y., 4, 63 (1985). Liljemark, W. F. et al., Infect. Immun. 26, 1104(1979). Reinholdt, J. et al., J. Dent. Res. 66, 492 (1987 ). This may bedone by agglutination of microorganisms, reduction of hydrophobicity,Magnusson, K. E., et al., Immunology 36, 439 (1979), or negative chargeand blockage of microbial adhesions. These anti-adherence effects areamplified by other factors such as secretory glycoproteins, continuousdesquamation of surface epithelium and floral competition. Abraham, S.N. et al., supra. Shedlofsky, S. et al., J. Infect. Dis. 129, 296(1974). For example, oral immunization against inactivated Vibriocholerae to induce a secretory immune response results in a 10-to 30-fold decrease in intestinal numbers.

Clinical experience with human peroral poliovirus vaccine and severalperoral or intranasal virus vaccines applied in veterinary medicineshows the sIgA plays a decisive role in protective effect by the mucosalimmune system against respiratory and enteric viral infections.Rusel-Jones, G. J. et al., Int. Arch. Allergy Appl. Immunol. 66, 316(1981). Ogra, P. L. et al., In J. Bienenstock (ed), Immunology of theLung and Upper Respiratory Tract. McGraw-Hill, N.Y., 242 (1984). theeffect of sIgA appears to be that of inhibiting the entry of virusesinto host cells rather than prevention of attachment. Taylor, H. P. etal., J. Exp. Med. 161, 198 (1985). Kilian, M. et al., Microbiol. Rev.52, 296 (1988).

B. General Overview of Plant Transformation

Various methods are known in the art to accomplish the genetictransformation by Agrobacterium species and transformation by directgene transfer.

1. Agrobactyerium-mediated Transformation

A. tumefaciens is the etiologic agent of crown gall, a disease of a widerange of dicotyledons and gymnosperms, DeCleene, M. et. al., Bot. Rev.42, 389 (1976), the results in the formation of tumors or galls in planttissue at the site the infection. Agrobactyerium, which normally infectsthe plant at wound sites, carries a large extrachromosomal elementcalled the Ti (tumor-inducing) plasmid.

Ti plasmids contain two regions required for tumorigenicity. One regionis the T-DNA (transferred-DNA) which is the DNA sequence that isultimately found stably transferred to plant genomic DNA. The otherregion required for tumorigenicity is the vir (virulence) region whichhas been implicated in the transfer mechanism. Although the vir regionis absolutely required for stable transformation, the vir DNA is notactually transferred to the infected plant. Chilton, M-D. et al., Cell11, 263 (1977), Thomashow, M. F. et al., Cell 19, 729 (1980).Transformation of plant cells mediated by infection with A. tumefaciensand subsequent transfer of the T-DNA alone have been well documented.See, for example, Bevan, M. W. et al., Int. Rev. Genet. 16, 357 (1982).

After several years of intense research in many laboratories, theAgrobactyerium system has been developed to permit routinetransformation of a variety of plant tissue See, for example. Schell, J.Et al., Bio/Technology 1, 175 (1983); Chilton, M-D, Scientific American248, 50 (1983). Representative tissues transformed in this mannerinclude tobacco, Barton, K. A. et al., Cell 32, 1033 (1983).Representative tissues transformed in this manner include tobacco,Barton, K. A. et al., Cell, 32, 1033 (1983); tomato, Fillatti, J. etal., Bio/Technology 5, 726 (1987); sunflower, Everett, N. P. et al.,.Bio/Technology 5, 1201 (1987); cotton, Umbeck, P. et al., Bio/Technology5, 263 (1987); rapeseed, Pua, E. C. et al., Bio/Technology 5, 815(1987); potato, Facciotti D. et al., Bio/Technology 3, 241 (1985);poplar, Pythoud, F. et al., Bio/Technology 5, 1323 (1987); and soybean,Hinchee, M. A. et al., Bio/Technology 6, 915 (1988).

Agrobacterium rhizogenes has also been used as a vector for planttransformation. That bacterium, which incites root hair formation inmany dicotyledonous plant species, carries a large extrachromosomalelement called an Ri (root-including) plasmid which functions in amanner analogous to the Ti plasmid of a A. tumefaciens. Transformationusing A. rhizogenes has developed analogously to that of A. tumefaciensand has been successfully utilized to transform, for example, alfalfa,Sukhapinda, K. et al., Plant Mol. Biol. 8, 209 (1987); Solanum nigrum L., Wei, Z-H, et al., Plant Cell Reports 5, 93 (1986); and, poplar,Pythoud, et al., supra.

2. Direct Gene Transfer

Several so-called direct gene transfer procedures have been developed totransform plants and plant tissues without the use of an Agrobacteriumintermediate. In the direct transformation of protoplasts the uptake ofexogenous genetic material into a protoplast may be enhanced by use of achemical agent or electric field. The exogenous material may then beintegrated into the nuclear genome. The early work was conducted in thedicot Nicotiana tabacum (tobacco) where it was shown that the foreignDNA was incorporated and transmitted to progency plants. Paszkowski, J.et al., EMBO J, 3, 2717 (1984); and Potrykus, I. et al., Mol. Gen.Genet. 199, 169 (1985).

Monocot protoplasts have also been transformed by this procedure: forexample, Triticum monococum, Lorz H. et al., Mol. Gen. Genet. 199, 178(1985); Lolium multiflorum (Italian ryegrass), Potrykus, I. et. al.,Mol. Gen. Genet 199, 183 (1985); maize, hodes, C., et al.,Bio/Technology 5, 56 (1988); and Black Mexican sweet corn, Fromm, M. etal., Nature 319, 719 (1986).

Introduction of DNA into protoplasts of N. tabacum is effected bytreatment of the protoplasts with an electric pulse in the presence ofthe appropriate DNA in a process called electroporation. From, M. E., inMethods in Enzymology, eds, Wu, R. and Grossman, L., Academic Press,Orlando, Fla., Volume 153, 307 (1987) and Shilliot, R. D. and Potrykus,I. in Methods in Enzymology, eds., Wu, R. And grossman, L., AcademicPress, Orlando, Fla., Volume 153, 283 (1987(. Protoplasts are isolatedand suspended in a mannitol solution. Supercoiled or circular plasmidDNA is added. The solution is mixed and subjected to a pulse of about400 V/cm at room temperature for less than 10 to 100 μsec. A reversiblephysical breakdown of the membrane occurs to permit DNA uptake into theprotoplasts.

DNA viruses have been used as gene vectors. A cauliflower mosaic viruscarrying a modified bacterial methotrexate-resistance gene was used toinfect a plant. The foreign gene was systematically spread in the plant.Brisson, N. et al., Nature 310, 511 (1984). The advantages of thissystem are the ease of infection, systematic spread within the plant,and multiple copies of the gene per cell.

Liposome fusion has also been shown to be a method for transformation ofplant cells. Protoplasts are brought together with liposomes carryingthe desired gene. As membranes merge, the foreign gene is transferred tothe protoplasts. Dehayes, A. et al., EMBO J. 4, 2731 (1985).

Polyethylene glycol (PEG) mediated transformation has been carried outin N. tabacum a dicot, and Lolium multiflorum, a monocot. It is achemical procedure of direct gene transfer based on synergisticinteraction between Mg²⁺, PEG, and possibly Ca²⁺. Negrutiu, R. et al.,Plant Mol. Biol. 8, 363 (1987).

Alternatively, exogenous DNA can be introduced into cells or protoplastsby microninjection. A solution of plasmid DNA is injected directly intothe cell with a finely pulled glass needle. In this manner, alfalfaprotoplasts have been transformed by a variety of plasmids, Reich, T. J.et al., Bio/Technology 4, 1001 (1986).

A more recently developed procedure for direct gene transfer involvedbombardment of cells by microprojectiles carrying DNA. Klein, T. M. etal, Nature 327, 70 (1987). In this procedure called particleacceleration, tungsten or gold particles coated with the exogenous DNAare accelerated toward the target cells. At lest transient expressionhas been achieved in onion. This procedure has been utilized tointroduce DNA into Black Mexican sweet corn cells in suspension cultureand maize immature embryos and laso into soybean protoplasts. Klein, T.M. et al., Bio/Technology 6, 559 (1988). McCabe, D. E. et al.,Bio/Technology 6, 923 (1988). Stably transformed cultures of maize andtobacco have been obtained by microprojectile bombardment. Klein, T. M.et al (1998), supra. Stably transformed soybean plants have beenobtained by this procedure. McCabe, D. E. et al., supra.

C. General Overview of Plant Regeneration

Just as there are a variety of methods for the transformation of planttissue, there are a variety of methods for the regeneration of plantsfrom plant tissue. The particular method of regeneration will depend onthe starting plant tissue and the particular plant species to beregenerated. In recent years, it has become possible to regenerate manyspecies of plants from callus tissue derived from plant explants. Theplants which can be regenerated from callus include monocots, such ascorn, rice, barley, wheat and rye, and dicots, such as sunflower,soybean, cotton, rapeseed and tobacco.

Regeneration of plants form tissue transformed with A. tumefaciens hasbeen demonstrated for several species of plants. These includesunflower, Everett, N. P. et al., supra; tomato, Fillatti, J. J. et al.,supra; white clover, White, D. W. R. et al., Plant Mol. Biol. 8, 461(1987); rapeseed, Pua, E-C. et al., supra; cotton, Umbeck, P. et al.,supra; tobacco, Horsch, R. B. et al., Science 225, 1229 (1985) andHererra-Estrella, L. et al., Nature 303, 209 (1983); and poplar, Pythoudet al., supra. The regeneration of alfalfa from tissue transformed withA. rhizogenes has been demonstrated by Sukhapinda, K. et al., supra.

Plant regeneration from protoplasts is a particularly useful technique.See Evans, D. A. et al., Handbook of Plant Cell Culture 1, 124 (1983).When a plant species can be regenerated from protoplasts, then directgene transfer procedures can be utilized, and transformation is notdependent on the use of A. tumefaciens. Regeneration of plants fromprotoplasts has been demonstrated for rice, Abdulah, R. et al.,Bio/Technology 4, 1987 (1987); tobacco, Potrykus, I. et al., supra;rapeseed, Kansha, et al., Plant Cell Reports 5, 101 (1986); potato,Tavazza, R. et al., Plant Cell Reports 5, 243 (1986); eggplant,Sihachaki, D. et al., Plant Cell, Tissue, Organ Culture 11, 179 (1987);cucumber, Jia, S-R., et al., J. Plant Physiol. 124, 393 (1986); poplar,Russel, J. A. et al., Plant Sci. 46, 133 (1986); corn, Rhodes, C. etal., supra; and the soybean McCabe, D. E. et al., supra.

D. Means for Inducing a Secretory Immune Response

The M cells overlying the Peyer's patches of the gut-associated lymphoidtissue (GALT) are capable of taking up a diversity of antigenic materialand particles (Sneller, M. C. and Strober, W., J. Inf. Dis. 154, 737(1986). Because of their abilities to take up latex and polystyrenespheres, charcoal, microcapsules and other soluble and particulatematter, it is possible to deliver a diversity of materials to the GALTindependent of any specific adhesive-type property of the material to bedelivered. In this case, antigen delivery to the GALT leads to ageneralized mucosal immune response with sIgA production against theantigen on all mucosal surfaces and by all secretory glands. One canalso stimulate a local secretory immune response by antigen delivery toa mucosal surface or to a secretory gland. The mechanisms(s) forgenerating such a localized secretory immune response is(are) poorlyunderstood. Recent evidence, Black, R. E. et al, Infect. Immun. 55, 1116(1987); Elson, C. O., in Curr. Top. Microbio. Immunol. 146, 29 (1989),indicate that the B subunit of cholera toxin when administered orallywith an antigen serves as an adjuvant to enhance the protective immuneresponse. It therefore follows, since the B subunit of cholera toxin aswell as of the E. coli heat-labile enterotoxin are capable of attachingto the GM-1 ganglioside of the intestinal epithelium and causingtranslocation across the epithelial membrane, that such pilot ortargeting proteins might be important in eliciting a local secretoryimmune response.

It is of course possible to consider fusing a gene for a givencolonization and/or virulence antigen to an N-terminal or C-terminalsequence specifying the B subunit of cholera toxin, the B subunit ofheat-labile enterotoxin, Yamamoto, T. et al. J. Biol. Chem. 259, 5037(1984), the PapG protein adhesion that specifically binds toα-D-galactopyranosyl-(1,4)-β-D- galactopyranoside, Lund, B. et al.,Proc. Natl. Acad. Sci. USA 84, 5898 (1987), or the invasions causingpenetration of bacteria through epithelial cell membranes as identifiedin and cloned from Yersinia pseudotuberculosis, Isberg, R. R., et al.Cell 50, 769 (1987), Shigella and Salmonella. Galan, J. et al., Poc.Natl. Acad. Sci. U.S.A. 86, 6383 (1989); Curtiss, R. III et al., in cur.Top. Microbiol. Immunol. 146, 35 (1989). In each case, it can beanticipated than the product of the gene fusion will be more readilytransported into cells of the intestinal mucosa and lead to enhancedlocal secretory immune responses. It is also possible that his form ofgene fusion would facilitate uptake and presentation of antigens to theGALT. The production of sIgA against a particular antigen can also befurther enhanced by the addition of orally-administered adjuvants, suchas microbial cell wall constituents Michalek, S. M. et al., in Curr.Top. Microbiol. Immunol. 146, 51 (1989);

It is therefore evident that stimulation of a specific sIgA response ofa both local and generalized nature can be achieved by oral immunizationwith purified proteins, Taubman, M. A. and D. J. Smith, in Curr. Top.Microbiol. Immunol. 146, 187 (1989), microencapsulated microbialproducts and viruses, Eldridge, J. H. et al., in Curr. Top. Microbiol.Immunol. 146, 59 (1989), whole-killed bacteria, Michalek et al., Science191, 1238 (1976), and by ingestion of live attenuated viruses, Cebra, etal., supra, and bacteria, Curtiss, R. III et al., in Proceedings of theTenth International Convocation on Immunology, 261. H. Kohler et al.,Eds., Longman Scientifc and Technical, Harlon, Essex, Great Britain(1987). The relative importance of the secretory immune system becomesapparent when one realizes that 80% of the antibody-secretary cells inthe body produce sIgA and that twice as much sIgA is secreted into thegastrointestinal tract than IgG is produced to enter the circulatorysystem each day, Brandtzaeg, P., in Curr. Top. Microbiol. Immunol. 146,13 (1989).

the Streptococcus mutans group of microorganisms constitute theprincipal etiologic agents of dental caries. Gibbons, R. J. et al., Ann.Rev. Med. 26, 121 (1975); Hamada, S. et al., Microbiol. Rev. 44, 331(1980). They colonize the tooth surface and remain there throughoutlife. Oral ingestion of killed S. mutans leads to the production of sIgAagainst S. mutans antigens in saliva, Michalek, S. M. et al., Science191, 1238 (1976); Mastecky, J. et. al., J. Clin. Invest. 61, 731 (1978)and this has been shown to be effective in preventing S. mutanscolonization on the teeth of rodents and primates and thereby preventinduction of caries. Michalek et al., supra; Challacombe, S. J. et al.,Arch. Oral Biol. 24, 917 (1980). Since sIgA must be present prior tocolonization to be effective, individuals immunized to produce sIgAagainst S. mutans colonization antigens after colonization to beeffective, will continue to be colonized with S. mutans unless thebacteria are mechanically removed during dental prophylaxis. Curtiss, R.III, in Curr. Top. Microbiol. Immunol. 118, 253 (1985). A diversity oftechniques are used to determine which surface constituents of apathogen are important for colonization and expression of virulence bythat pathogen. Thus mutants can be isolated and tested for ability tocolonize or cause disease. Gene cloning can be used to produce a geneproduct in a heterologous microorganism. The expressed gene product canbe used to immunize animals to see whether colonization and/or virulenceby the pathogen is inhibited. Based on such studies, scientists caninfer relative importance to various colonization and virulence antigensand thereby choose those that are appropriate to use in vaccinecompositions so as to immunize human or other animal hosts and preventcolonization and infection by the pathogen. Such studies have beenperformed with the S. mutans group of microorganisms to demonstrate thecritical importance of the surface protein antigen A (SpaA; also knownas antigen I/II, B, and P1), glucosyltransferases, dextranase andglucan-binding proteins. Curtiss, 1985 supra.

The surface protein antigen A (SpaA) constitutes a major protein antigenon the surface of S. mutans. Curtiss, R. III, et al., in StreptococcalGenetics, Ferretti, J. J. et al., Ed. American Society for Microbiology,Washington, D.C. pp. 212-216 (1987). The spaA gene has been cloned,Holt, R. G. et al., Infect. Immun. 38, 147 (1982), partially sequencedand the major antigenic determinants mapped. It is known that mice andhumans intentionally or naturally immunized by oral ingestion of S.mutans produce sIgA in saliva against the SpaA protein. It isfurthermore known that immunization of monkeys with antigen I/II (whichis essentially immunologically identical to SpaA, Holt et al., supra)yields protective immunity against S. mutans colonization and S.mutans-induced dental caries, Russell, M. W. et al. Immunol. 40, 97(1980).

Invasive Salmonella, such as S. typhimurium and S. typhi constitute theetiologic agents for typhoid fever in mice and humans, respectively.They gain access to deep tissues following oral ingestion by attachingto, invading, and proliferating in the GALT. Carter and Collins J. Exp.Med. 139, 1189 (1974). Salmonella can be rendered avirulent so as not toinduce disease by introducing mutations in known genes. Germanier, R. etal., Infect. Immun. 4, 663 (1971); Germanier, R. et al., J. Infect. Dis.131, 553 (1975); Hoiseth and Stocker, Nature 291, 238 (1981); Curtiss,et al., Infect. Immun. 55, 3035 (1987). Such mutants are immunogenicwhen administered orally and retain their tissue tropism for the GALT.Curtiss, R. III, J. Dent. Res. 65, 1034 (1986); Curtiss, R. III et al.,in Proceedings of the Tenth International Convocation on Immunology,261. H. Kohler et al., Eds., Longman Scientific and Technical, Harlon,Essex, Great Britain (1987); Curtiss, R. III, et al., Infect. Immun. 55,3035 (1987).

A number of S. typhimurium and S. typhi strains which posses variousdeletion mutation rendering them avirulent have been constructed withthe ability to produce colonization and/or virulence antigens fromseveral pathogens. Oral immunization leads to production of sIgA and IgGresponses against the expressed antigen. Formal, S. B. et al., Infect.Immun. 34, 746 (1981); Stevenson, G. et al., FEMS Microbiol. Lett. 28,317 (1985); Clements, J. D. et al., Infect. Immun. 53, 685 (1986);Maskell, D. et al., in Vaccines 86, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. pp. 213-217 (1986). Recombinant avirulent Salmonellaexpressing the S. mutans SpaA and glucosyltransferase proteins have beenconstructed. Curtiss et al., in: The Secretory Immune System, J. R.McGhee and J. Mestecky, Eds., Ann. N.Y. Acad. Sci. 409, 688 (1983);Curtiss supra (1986); Curtiss et al., supra (1987); Curtiss et al.,Vaccine 6, 155 (1988). Secretory antibodies (sIgA) against SpaA havebeen produced in saliva following oral immunization with avirulentSalmonella strains expressing the S. mutans SpaA protein, Curtiss, R.III et al., in Mol. Microbiol. Immunol of Streptococcus mutans, Hamada,S. et al., Eds., Elsevier, NY, pp. 173-180 (1986); Katz, J. et al., inRecent Advances in Mucosal Immunology, Part B, mestecky, J. et al., Ed.,Plenum Publishing Corp. pp 1741-1747 (1987); Curtiss et al., 1987,supra.

SUMMARY OF THE INVENTION

The present invention is directed to transgenic plants which contain DNAsequences which code for a colonization antigen, a virulence antigen,antigenic determinants thereof or fusion proteins thereof of pathogenicmicroorganisms. The present invention is further directed tocompositions useful for stimulating secretory immunity in human andanimals. The present invention is also directed to methods for makingthe compositions and producing transgenic plants and to methods forstimulating secretory immunity.

More specifically, the present invention is directed to transgenicplants which are capable of expressing a colonization antigen, avirulence antigen or antigenic determinants thereof of pathogenicmicroorganisms. The transgenic plants are useful for orally immunizinghumans and animals to elicit a secretory immune response in the human oranimals to inhibit colonization and/or invasion through a mucosalsurface by said pathogenic microorganism.

The transgenic plants are produced by transforming plants with a planttransformation vector which contains at least one DNA sequence whichcodes for an antigen of a pathogenic microorganism. The antigen may be acolonization antigen, a virulence antigen, an antigenic determinant ofeither antigen or a fusion protein containing either antigen ordeterminant. In addition to the antigen or antigenic determinant, thefusion protein may contain a polypeptide which stabilizes and/orenhances the activity of the antigen. The fusion protein may also be oneor more antigens.

The plant transformation vectors are prepared by inserting one or moreDNA sequences coding for the antigen of interest into a vector suitablefor the transformation of plants. The vectors may be used for directgene transfer or for agroinfection to insert the DNA sequences into thedesired plants. The DNA sequences may be natural or synthetic and maycomprise an entire gene or a fragment of a gene which codes for theantigen.

The compositions useful for eliciting a secretory immune response may betransgenic plant itself or material derived from the plant. For example,the transgenic plant could be ingested directly by humans or animals orit could be processed to make a food product which is ingested by humansor animals. The compositions are useful for immunizing humans or animalsagainst the pathogenic microorganisms to which the antigens correspond.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the construction of the plasmid pSUN450.

FIG. 2 illustrates the construction of the plasmid pSUN470.

FIG. 3 illustrates the construction of the plasmid pSUN221.

FIG. 4 illustrates the construction of the plasmid pSUN473.

FIG. 5 illustrates the construction of the plasmid pSUN475.

FIG. 6A-6C illustrates the construction of the plasmids pSUN339,pSUN340, pSUN341, pSUN342, pSUN343.

FIG. 7 illustrates the construction of the plasmids pSUN387, pSUN390,pSUN391, pSUN392, pSUN393 and pSUN394.

FIG. 8A-8C depicts the entire 4,643 base pair nucleotide sequence ofpSUN387.

FIG. 9 illustrates the results of a Western blot analysis of SpaAprotein synthesis detected by rabbit anti-SpaA sera in E. coli DH5α(lane 2) and in E. coli DHα containing plasmids pSUN341 (lane 3),pSUN342 (lane 4), pSUN343 (lane 5), pSUN344 (similar or identical topSUN343; lane 6), pSUN345 (similar or identical to pSUN343; lane 7) andpSUN346 (similar or identical to pSUN341; lane 8). Prestained molecularweight markers are included in lane 1.

FIG. 10 illustrates the results of a Western blot analysis of SpaAprotein synthesis as revealed by reaction with rabbit anit-SpaA sera inE. coli X2991 containing pYA177, pYA178, pYA179 and pYA180 and in E.coli DH5α containing pSUN390, pSUN391, pSUN392, pSUN393, and pSUN394.Lane 1 contains prestained molecular weight standards.

FIG. 11 illustrates densitometric quantitation of the amount of SpaAprotein synthesized by transgenic tobacco plants.

FIG. 12 illustrates the results of a Western blot analysis of a SDSpolyacrylamide gel which compares samples of tobacco which produce SpaAprotein to samples of tobacco which do not produce SpaA protein and alsocompares fresh samples, samples lyophilized and stored at -20° C.,samples stored at room temperature and samples mixed with commercialsmouse meal.

Lane 1 contains pre-stained molecular weight standard.

Lane 2 contains 150 μg protein from a cell extract of tobacco notproducing SpaA.

Lane 3 contains 150 μg protein from a cell extant of tobacco producingSpaA.

Lane 4 contains 150 μg protein from a cell extract of lyophilizedtobacco which does not produce SpaA, and which was stored at -20° C. for13 days.

Lane 5 contains 150 μg protein from a cell extract of lyophilizedtobacco which produces SpaA, and which was stored at -20° C. for 13days.

Lane 6 contains 150 μg protein from a cell extract of lyophilizedtobacco which does not produce SpaA, and which was stored at roomtemperature for 13 days.

Lane 7 contains 150 μg protein from a cell extract of lyophilizedtobacco which produces SpaA, and which was stored at room temperaturefor 13 days.

Lane 8 contains 300 μg protein extract of 1:1 mixture of mouse meal tolyophilized tobacco which does not produce SpaA. The tobacco was storedat room temperature for 13 days.

Lane 9 contains 300 μg of protein extract of 1:1 mixture of mouse mealto lyophilized tobacco which produces SpaA. The tobacco was stored atroom temperature for 13 days.

Lane 10 contains 150 μg protein extract of mouse meal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes (a) plants, seeds, and plant tissuecapable of expressing an antigen selected from the group of colonizationand/or virulence antigens, and/or antigenic determinants thereof and/orfusion proteins of the antigens or determinants of pathogens; (b)compositions useful for the stimulation of a secretory immune responsein an human or other animal; (c) methods for stimulating a secretoryimmune response in humans and other animals so as to inhibitcolonization and/or invasion through mucosal surfaces by pathogens; (d)unique vectors containing DNA sequences coding for colonization orvirulence antigens; and (e) a method of producing colonization orvirulence antigens of pathogenic microorganisms in plants.

In order to provide a clear and consistent understanding of thespecification an the claims, including the scope given to such terms,the following definitions are provided:

Antigen: A macromolecule which is capable of stimulating the productionof antibodies upon introduction into a human or other animal. As usedherein, antigen shall mean an antigen per se, and antigenic determinantof the antigen, or a fusion protein containing the antigen or antigenicdeterminant.

Antigenic Determinant: A small chemical complex that determines thespecificity of an antigen-antibody reaction. Colonization and/orvirulence antigens of a pathogen contain one or more antigenicdeterminants.

Colonization or Virulence Antigens: Antigens on the surface of apathogenic microorganism that are associated with the ability of themicroorganism to colonize or invade its host. Discussions and claims mayrefer to colonization or virulence antigens or antigenic determinantsthereof. A pathogen may contain antigens of either colonization orvirulence or both and one or more DNA sequences for each or both may betransferred to a vector and used to transform a plant such that itexpresses the antigen or antigens.

Chimeric Sequence or Gene: A DNA sequence containing at least twoheterologous parts, e.g., parts derived from, or having substantialsequence homology to, pre-existing DNA sequences which are notassociated in their pre-existing states. The pre-existing DNA sequencesmay be of natural or synthetic origin.

Coding DNA Sequence: A DNA sequence from which the information formaking a peptide molecule, mRNA or tRNA are transcribed. A DNA sequencemay be a gene, combination or genes or a gene fragment.

Food: Food or foodstuff or feedstuff is a plant or any material obtainedfrom a plant which is ingested by humans and other animals. This term isintended to include raw plant material which may be fed directly tohumans and other animals or any processed plant material which is fed tohumans and other animals. Materials obtained from a plant are intendedto include any component of a plant which is eventually ingested by ahuman or other animal.

Foreign DNA: DNA which is exogenous to or not naturally found in themicroorganism or plants to be transformed. Such foreign DNA includesviral, prokaryotic, and eukaryotic DNA, and may be naturally occurringDNA, chemically synthesized DNA, cDNA, mutated DNA or any combination ofthe same. The foreign DNA of the present invention is derived from orhas substantial sequence homology to DNA of pathogenic microorganismsand viruses.

Gene: A discrete chromosomal region which is responsible for a discretecellular product.

Microorganism: A member of one of the following classes: bacteria,fungi, protozoa or viruses.

Plant Tissue: Any tissue of a plant in plant or in culture. This termincludes, but is not limited to, whole plants, plant cells, plantorgans, plant seed, protoplasts, callus, cell cultures and any group ofplant cells organized into structural and/or functional units. The useof this term in conjunction with, or in the absence of, any specifictype of plant tissue as listed above or otherwise embraced by thisdefinition is not intended to be exclusive of any other type of planttissue.

Plant Transformation Vector: A plasmid or viral vector that is capableof transforming plant tissue such that the plant tissue contains andexpresses DNA not pre-existing in the plant tissues.

Pre-existing DNA Sequence: A DNA sequence that exists prior to its use,in toto or in part, in a product or method according to the presentinvention. While such pre-existence typically reflects a natural origin,pre-existing sequences may be of synthetic or other origin.

Secretory Immune Response: The formation and production of secretory IgAantibodies in secretions that bathe the mucosal surface of humans andother animals and in secretions from secretory glands. An agent whichcauses the formation and production of such antibodies is considered tostimulate secretory immunity or to elicit a secretory immune response.Secretory immunity is also sometimes referred to as mucosal immunity.

Substantial Sequence Homology: Substantial functional and/or structuralequivalence between sequences of nucleotides or amino acids. Functionaland/or structural differences between sequences having substantialsequence homology will be de minimus.

Transgenic Plant: A plant which contains and expresses DNA that was notpre-existing in the plant prior to the introduction of the DNA into theplant.

Colonization and/or Virulence Antigens of Escherichia coli

Effective immunity against the enterotoxic E. coli that colonize pigs,calves, and humans can be achieved by including plant materialsexpressing the K88 pilus colonization antigen for the swine feed, K99pilus colonization antigen for the calf feed, and the CFA piluscolonization antigen for humans. Plant material containing the B subunitof the E. coli enterotoxin can be included in feed for humans, calves,and swine to serve as an adjuvant to enhance the immune response to thepilus colonization antigens but also to induce protective immunityagainst the enterotoxin which is produced by most enterotoxigenicstrains of E. coli infecting swine, calves and humans. Since one couldmake a diversity of blends, it would be possible to immunize against alarge number of pathogens at the same time. Continual feeding of thefood, at the appropriate time, could be used not only to induceprotective immunity in young animals but also for immunization of adultfemales of the species to permit transmission of effective immunity tothe offspring either through eggs, by placental transfer, or incolostrum and milk.

Heat-Labile Toxin B Subunit

The nucleotide sequence of the gene for the heat-labile enterotoxin ofE. coli has been determine. Yamamoto, T. and Yokota, T., J. Bacteriol.155, 728 (1983). The EcoRI-HindIII DNA fragment containing the entire Bsubunit gene can be inserted into the plant transformation vectorpSUN387 (see FIG. 7) by methods standard in the art and further modifiedusing either oligonucleotide synthesis or the restriction enzymes NspBiior MaeI, which cleave the nucleotide sequence either seven amino acidsor zero amino acids from the C-terminal end to permit insertion of amultiple cloning site to facilitate making a great diversity of fusiongene products leading to the production of fusion proteins with theheat-labile toxin B subunit (LT-B) as the N-terminus sequence.

It is thus possible to insert the LT-B sequence in a planttransformation vector and have it expressed in a suitable plant species.Induction of sIgA against LT-B will block uptake or the intact LT toxinand thereby decrease the severity of the diarrhea associated withenterotoxigenic E. coli infection. If the plant also produces K99 K88 orone of the other pilus adhesive antigens, the induction of a sIgAresponse should also inhibit colonization by the enterotoxigenic E. coliand thus greatly diminish diarrhea.

E. coli Pap Pili genes

Pap pili genes or uropathogenic E. coli including genes for the pilusadhesion have been cloned, Lund, B., et al. J. Bacteriol 162, 1293(1985) and have subsequently been sequenced. Uropathogenic E. coli mayexpress several different pilus adhesins and cloned genes expressingseveral different pilus types are available, Clegg, S. Infect. Immun.38, 739 (1982); Van Die, I., et al. FEMS Microbiol. Lett. 19, 77 (1983);Normark, S., et al., Infect. Immun. 41, 942 (1983).

E. coli K99 Pilus Antigen

K99 pilus antigen is expressed by enterotoxigenic E. coli strainscausing scours in calves. The gene for the K99 pilus antigen has beencloned and expressed, van Embden, J. D. A., et al. Infect. Immun. 29,1125 (1980); deGraaf, F. K., et al., Infect. Immun. 43, 508 (1984) andsequenced, Roosendahl, E., et al., FESM Microbiol. Lett. 22, 253 (1984).It is therefore straightforward to insert it into a plant transformationvector. Induction of sIgA against K99 pili blocks colonization in thecalf intestine and thereby prevents scours.

E. coli K88 Pilus Antigen

K88 pilus antigen is expressed by enterotoxigenic E. coli strainscausing severe diarrhea disease in pigs. The gene for the K88 pilusantigen necessary for intestinal colonization has been cloned, Mooi, F.R., EL et al., Nuc. Acids Res. 6, 849 (1979), Kehoe, M. et al., J.Bacteriol. 155, 1071 (1983) and sequenced, Gaastra, W., et al., FEMSMicrobiol. Lett. 12, 41 (1981). It is therefore possible to insert thissequence into a plant transformation vector in such a way as to causeits synthesis in plants.

Genes for other pilus adhesins that permit colonization ofenterotoxigenic and enteropathogenic E. coli in humans and in otheranimal hosts have been identified and in some cases cloned andsequenced, see Mooi, F. R. and deGraaf, F. K., Curr. Top. Microbiol.Immunol. 118, 119 (1985); Kaper, J. B. and M. M. Levine, Vaccine 6, 197(1988).

Colonization and/or Virulence Antigens of Streptococcus mutans

Protein associated with the surface of S. mutans include the surfaceprotein antigen A (SpaA), glucosyltransferase B (GtfB), dextranase,glucosyltransferase C (GtfC), and glucan binding proteins. S. mutansserotype α surface protein antigen A (SpaA)

The spaA gene from S. mutans serotype g strain UAB90 was cloned on acosmid vector in E. coli. Holt, et al., 1983, supra. The protein isessential for the initial colonization of the tooth surface and itsabsence precludes colonization of germ free rats (Curtiss, et al., 1987asupra; 1987b supra). The spaA gene has been subcloned, the majorantigenic determinants of the protein determined and these regions ofthe gene sequenced.

S. mutans glucosyltransferase B

The S. mutans glucosyltransferase B is encoded by the gtfB gene andsynthesizes water-insoluble glucan polymers and free fructose fromsucrose. The gene has been cloned and sequenced by Shiroza, T. et al. J.Bacteriol. 169, 4263 (1987). The plasmid pSU20 (9.3 kb) contains a 6.5kb PstI fragment encoding the 165,800 kilodalton (kDa) GtfB protein.Based on the known nucleotide sequence and the location of the ATG startcodon, the coding sequence is inserted into a plant transformationvector using conventional techniques.

S. sobrinus (S. mutans serotype g) dextranase gene

The pYA902 cosmid clone expresses S. sobrinus dextranase, Barret, J. F.et al., Infect. Immun. 55, 792 802 (1987), and Jacobs, W. R. et al.,Infect. Immun. 52, 101 (1986). A partial PvuII digest of pYA902 DNAgenerated a series of plasmids with all or portions of the dextranasegene. pYA993 is a 5.45 kb plasmid expressing a slightly truncateddextranase of 110 kDa. A 2.6 kb PvuII fragment containing all of thedextranase coding sequence in pYA993 has been cloned in the correctorientation into the SmaI site of pUC8 by blunt-end ligation. Thisfragment has the dextranase ATG start codon but lacks the dextranasepromoter. Thus it can readily be inserted into a plant transformationvector either to be directly expressed under the control of a plantpromoter or as a tandem fusion construction fused to the C-terminal endof the spaA coding sequence, for example.

Plant Transformation Vectors

The vectors of the present invention are vectors which contain DNAcoding for colonization and/or virulence antigens and are capable oftransforming plants. Foreign DNA is DNA which is exogenous to or notnaturally found in the organism to be transformed. It can be insertedinto cloning vectors to transform plants. The foreign DNA of the presentinvention is derived from or has substantial sequence homology to DNA ofpathogenic microorganisms and viruses. The vectors of the presentinvention are produced by standard techniques. However, the vectorproduced will depend on which type of transformation and which speciesof plant is being transformed. For example, if plant protoplasts arebeing transformed, the vector can be a Ti plasmid-derived vector or anyvector which can be introduced by direct gene transfer means into theprotoplasts. If a plant or plant organ or part thereof if beingtransformed, then the vector must be capable of transforming this typeof tissue. In this instance, the novel plant transformation vector willlikely be based on a Ti plasmid-derived vector, although vectors usefulfor microprojectile transformation can also be used. Appropriate vectorswhich can be utilized as starting materials are known in the art.Suitable vectors for transforming plant tissue have been described bydeFramond, A. et al., Bio/Technology 1, 263 (1983); An, G. et al., EMBOJ. 4, 277 (1985); Potrykus, I. et al., supra; Rothsetin S. J. et al.,Gene 53, 153 (1987), as well as the other vectors described in thereferences discussed above. In addition to these vectors, many othershave been produced in the art which are suitable for use in the presentinvention.

The construction of the vectors can be performed in a suitable host, forexample, E. coli. Suitable E. coli strains include but are not limitedto HB101, JM83, DH1, DH5α, LE392 and the like. If the vectors are usedin a direct gene transfer or a micro-injection technique, they can beused directly. In certain instances it may be preferable to linearizethe vector before use. If the vectors are to be used in an A.tumefaciens host, then the vector must first be transferred to theappropriate strain. This transfer is accomplished by conventionaltechniques, including biparental mating, Simon, R. et al.,Bio/Technology 1, 74 (1983); triparental mating, Ditta, G. et al., Proc.Natl. Acad. Sci. USA 77, 7347 (1980) or transformation; Holster, M. etal., Mol. Gen. Genet. 163, 181 (1978). Suitable strains of A.tumefaciens include but are not limited to LBA4404.

The vectors of the present invention contain DNA sequences encodingcolonization or virulence antigens from a variety of pathogens known tocause diseases in humans and other animals. While the followingdescription and many of the examples are directed to DNA sequences foundnaturally in pathogenic bacteria, this discussion applied equally tosuch sequences which occur and can be cloned from, viral, fungal andparasitic pathogens. Of course, DNA sequences derived by synthesis toencode colonization and/or virulence antigens or parts thereof, aresimilarly embraced.

A DNA sequence coding for a colonization or virulence antigen or a partof the antigen of a pathogen is obtained by conventional means andinserted into any vector suitable for the transformation of plants. Forexample, the DNA sequence can be isolated from a gene bank of genomicclones. Alternatively, the DNA sequence can be prepared by reversetranscription. The vectors are then introduced into plant cells by avariety of known techniques which give rise to transformed cells,tissues and plants.

The DNA sequence can be chemically synthesized if the amino acidsequence of the colonization or virulence antigen or part thereof isknown. Several prior art methods can be utilized to determine the aminoacid sequence of the colonization or virulence antigen. A part of theamino acid sequence can be determined and used to prepare a probe forreverse transcriptions.

The DNA sequence can contain a coding sequence for the specific aminoacid sequence of the colonization or virulence antigen, or for one ormore of its antigenic determinants. The DNA sequence can also containadditional coding sequences which code for all or part of a proteinwhich contains the colonization or virulence antigen.

The DNA sequence encoding the colonization or virulence antigen or partthereof of a pathogenic microorganism is inserted into an appropriatevector in such a manner that the colonization or virulence antigen iscorrectly expressed. In other words, the DNA sequence is positioned inthe proper orientation and reading frame so that the correct amino acidsequence is produced upon expression of the DNA sequence in planttissue. In accordance with conventional techniques, a chimeric DNAsequence is generally constructed which contains a promoter operable inplant tissue and the DNA sequence coding for the colonization orvirulence antigen. The chimeric DNA sequence may further contain 3'non-coding sequences operable in plant tissue. The chimeric DNA sequencemay further contain a coding sequence for a polypeptide other than theprotein containing the colonization or virulence antigen such that afusion protein is produced upon expression. The chimeric DNA sequencecan be prepared in situ within a suitable vector by inserting the DNAsequence coding for the colonization or virulence antigen into arestriction site of a known plant transformation vector. Alternatively,the chimeric gene could be first constructed and then inserted into avector to produce a plant transformation vector.

A colonization or virulence antigen or part thereof can be modified toincrease its resistance to proteolytic breakdown. To do this, it ispossible to genetically engineer a fusion construct between acolonization or virulence antigen and a peptide that is completelyresistant to intestinal proteases and which acts as an adjuvant oforally administered antigens. The LT-B subunit has both of thesecharacteristics. Other peptides include the B subunit of choleratoxin(CT-B), PapG protein adhesion and the like discussed above. The fusionconstruct is prepared by conventional techniques.

Plant Transformation

The cells of plants are transformed with the vectors described above byany technique known in the art, including those described in thereferences discussed above and by techniques described in detail in theexamples which follow. These techniques include but are not limited todirect infection or co-cultivation of plants or plant tissue with A.tumefaciens. A very suitable technique is the leaf disk transformationdescribed by Horsh, R. B. et al., Science 225, 1229 (1985).

Alternatively, the vector can be transferred directly, for example byelectroporation, by microinjection, by microprojectiles or bytransformation of protoplasts in the presence of polyethylene glycol(PEG), calcium chloride or in an electric field.

Following transformation, the transformed cell or plant tissue isselected or screened by conventional techniques. The transformed cell orplant tissue containing the chimeric DNA sequence discussed above isthen regenerated by known procedures, including those described in thereference discussed above and in the examples which follow for bothmonocot and dicot plants. The species which can be regenerated by thesetechniques include, but are not limited to, maize, sunflower, rapeseed,clover, tobacco, cotton, alfalfa, rice, potato, eggplant, cucumber andsoybean. The regenerated plants are screened for transformation bystandard methods. Progency of the regenerated plants are screened andselected for the continued presence of the integrated DNA sequence inoder to develop improved plant and seed lines. The DNA sequence can bemoved into other genetic lines by a variety of techniques, includingclassical breeding, protoplast fusion, nucelar transfer an chromosometransfer.

Compositions for Inducing Immunity

The level of expression of an antigen can often be affected by the siteof insertion into the vector. The quantity of a colonization orvirulence antigen expressed in transgenic plants can be also optimizedby retransformation with suitable vectors to increase the number of genecopies for the colonization and/or virulence antigen. Production of SpaAprotein can be maximized by retransformation in at least three differentways. Vectors describe above, constructs of vectors with enhancedpromoter efficiency, or vectors carrying multiple copies of the spaAgene sequence or the sequence for a SpaA antigenic determinant can beinserted into plants already carrying spaA genetic material.

A large number of regenerated plants should be examined for productionof colonization or virulence antigens. Those plants yielding the highestlevel of stable production of colonization or virulence antigens areselected. If the turnover rate of the colonization or virulence antigenis unacceptably high, the protein could be modified by a variety ofprocedures to enhance the stability of the protein in plants (i.e.,removal or alteration of protease cleavage sites by site-directedmutagenesis of the DNA sequence encoding the antigen). The genespecifying the protein could be engineered so that the protein isintroduced as a storage protein in seed and thereby ensure high levelsof stable production. This would be most practical in soybean and cerealgrains for example.

In order to be an effective immunogen a colonization or virulenceantigen expressed by a plant must be sufficiently stable to withstandfood processing and digestion.

The plant material may be fed directly to a human or other animal orprocessed into food by means that will not denature protein. Forexample, transgenic plants, such as alfalfa or maize, containing adesired colonization or virulence antigen could be fed directly tohumans or to other animals such as cattle. If the colonization orvirulence antigen was from a colonization factor of enteropathogenic orenterotoxigenic E. coli, secretory immunity to scours can be produced inthe cattle. Similarly, the seeds, of a variety of transgenic plantsexpressing colonization or virulence factors of a pathogenicmicroorganism could be directly eaten by humans in order to elicit asecretory immune response against it.

Alternatively, the transgenic plant can be processed by conventionaltechniques to produce food for humans and other animals. For example,transgenic maize can be processed to produce cornmeal which can be fedto animals or used to prepare foods for humans.

It is conceivable in some instance that a colonization or virulenceantigen might not be readily denaturable, therefore, in some cases,cooking of a foodstuff might not destroy immunogenicity. This is truewith regard to the SpaA protein which retains its immunoreactivity afterdenaturation by boiling or by treatment with ionic detergents. On theother hand, other colonization antigens or virulence antigens might notbe so resilient to denaturation. In some cases increased stability ofthe colonization or virulence antigen to denaturation can be achieved byfusing the antigen to a polypeptide that inhibits denaturation orfosters spontaneous renaturation under suitable conditions.

Stability of Colonization and Virulence Antigens

The quantity, stability and immunogenicity of a major colonizationand/or virulence antigen of a pathogenic microorganism in transformedplants may be evaluated by means that are well known, particularlyimmunological means. These variables can be measured in transformedprotoplasts and callus, and in the roots, stems, leaves and seed ofmature plants.

Colonization and/or virulence antigens specified by S. mutans or E. coliDNA in plant vectors and expressed in recombinant E. coli and othersuitable microorganisms can be tested for stability after feeding. Aculture of a microorganism which expresses a known colonization and/orvirulence antigen is killed by known methods such as heat or radiation.It is then added to a known animal food such as commercially availablemouse meal and subjected to food processing. Protein from the enhancedand processed mouse meal may be analyzed for quantity of thecolonization and/or virulence antigen before feeding and at variousstages of digestion after feeding. Analysis may be carried out by avariety of known methods including but not limited to western blotanalysis following sodium dodecylsulfate (SDS) polyacrylamide gelelectrophoesis, immunoprecipitation and enzyme linked immunosorbantassay (ELISA). The quantity of the antigen at various stages ofdigestion may be compared to the quantity before ingestion.

Immunological Response Following Oral Ingestion of Colonization andVirulence Antigens

Immunogenicity of the antigen is analyzed as well. Recombinant E. coliexpressing colonization and virulence antigens are evaluated for theability upon feeding to elicit a secretory immune response which isdependent upon the ability of the colonization and the virulenceantigens to survive through the intestinal tract without destruction oftheir immunogenicity by intestinal enzymes. Plant material enhanced withS. mutans, recombinant E. coli or other suitable microorganisms whichhave been killed by heat or radiation, is subject to food processing. Itmay then be stored dry or frozen. Transgenic plants are also processedand either fed to animals or mixed with animal feed and theimmunogenicity is determined by quantitative sIgA against thecolonization or virulence antigen in saliva or in intestinal washesusing Enzyme Linked Immunosorbent assay (ELISA).

Recombinant DNA Methods Used In the Examples Below

DNA manipulations were carried out using enzymes in accordance with themanufacturers' recommended procedures unless indicated otherwise. Allenzymes were obtained from New England BioLabs or Bethesda ResearchLaboratories (BRL). All vector constructions were carried out in E. coliDH1, JM83 or DH5α unless indicated otherwise. The vectors wereintroduced into strains of E. coli different from the constructionstrains using conventional techniques. DNA isolations and E. colitransformations were conducted in accordance with Hanahan et al., J.Mol. Biol. 166, 557 (1983). Blunt-end ligations in 15% polyethyleneglycol (PEG) were performed in accordance with Livak, Anal. Biochem.152, 66 (1986). Additional techniques are described in Maniatis, T. etal., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1988), Methods in Enzymology Vol68 (1979), Vol 100 (1983), Vol 101 (1983), Vol 118 (1986) and Vol152-154 (1987); and Plant Molecular Biology: Manual, Gelvin, SB andSchilpeoort, RA, eds., Kluwer Academic Publishers, Dodrecht (1988).

Example 1 I. Vector Constructions

Vectors useful for expressibly transforming plants with DNA sequencesencoding colonization or virulence antigens are pSUN341 and pSUN343.Extensive information has been included in example 1 in order to enablethe construction of these vectors from starting materials that arewidely known and generally available. The extensive informationavailable herein will enable the construction of similar vectors fromother starting material.

A. Construction of Plasmid Vectos pSUN341 and pSUN343

1. Construction of pSUN450

The plasmid pSUN214 (ATCC 67470) was digested with PstI and HindIII. The1.6 kb fragment was isolated containing the gene for chloramphenicalacetyl transferease (CAT) and the 3'-NOPS (nopaline synthase) sequenceto provide a site for PolyA addition required for eukaryotic geneexpression. The plasmid pUC18 was digested with PstI and HindIII andligated with the isolated fragment. The resulting plasmid pSUN218 wasisolated.

The plasmid pSUN218 was digested with SmaI and treated with calfintestinal alkaline phosphatase. The plasmid pCE101, Guilley, H. et al.,Cell 30, 763 (1982), was obtained from K. Richards and digested withHphI. The fragment containing the 35S promoter of cauliflower mosaicvirus, a sequence which permits transcription in plant cells wasisolated and treated with T4 DNA polymerase. This fragment was blunt-endligated in 15% PEG to the treated pSUN218 to produce the plasmidpSUN444.

The plasmid pSUN204 (ATCC 67469) was digested with HindIII and thepartially digested with PstI. The larger 1.6 kb APTII-3'-NOPS (nopalinesynthase) sequence to provide a site of PolyA addition required foreukaryotic gene expression. The plasmid pUC18 was digested with PstI andHindIII and ligated with the isolated fragment. The resulting plasmidpSUN218 was isolated.

The plasmid pSUN218 was digested with SmaI and treated with calfintestinal alkaline phasphatase. The plasmid pCE101, Guilley, H. et al.,Cell 30, 763 (1982), was obtained from K. Richards and digested withHphI. The fragment containing the 35S promoter of cauliflower mosaicvirus, a sequence which permits transcription in plant cells wasisolated and treated with T4 DNA polymerase. This fragment was blunt-endligated in 15% PEG to the treated pSUN218 to produce the plasmidpSUN444.

The plasmid pSUN204 (ATCC 67469) was digested with HindII and thenpartially digested with PstI. The larger 1.6 kb APTII-3'-NOPS containingfragment was isolated. The APTII gene confers resistance to theantibiotics kanamycin, neomycin and G418 and as such provides a usefultransformation, selection determinant in plants. The plasmid pUC18 wasdigested with PstI and HindIII and ligated to the APT II-3'-NOPSfragment. The resulting plasmid was identified as pSUN219.

The plasmid pSUN219 was digested with SalI and treated with the Klenowfragment of DNA polymerase I. The DNA was then digested with HindIII andthe fragment containing the APT II-3'-NOPS sequences was isolated. Theplasmid pSUN444 was digested with BamHI and treated with the Klenowfragment of DNA polymerase I. The treated vector was then digested withHindIII. The 3.5 kb large fragment was isolated and ligated to the APTII-3'-NOPS containing fragment from pSUN219. The resulting plasmid wasidentified as pSUN450. The construction and partial map of pSUN450 areillustrated in FIG. 1.

2. Construction of pSUN470

The plasmid pSUN450 was digested with HindIII and treated with theKlenow fragment of DNA polymerase I. The treated vector was thendigested with KpnI, and the 2.5 kb fragment containing the 35S promoter,APT II and 3'-NOPS sequences was isolated. The plasmid pGA470 (obtainedfrom G. An) was digested with SalI and EcoRI. A 500 fragment containingthe left border (B_(L)) sequence was isolated and ligated into theExcoriate of pUC18. The vector pSUN402 was identified and isolated.

The plasmid pSUN402 was digested with EcoRI and treated with the Klenowfragment of DNA polymerase I. The treated vector was the digested withKpnI and ligated to the fragment isolated from pSUN450. The plasmidpSUN470 was identified. The construction and partial map of pSUN470 areillustrated in FIG. 2. pSUN470 contains the multiple cloning site (MCS)of pUC18 including restriction sites from KpnI through HindIII.

3. Construction of pSUN221

The plasmid pSUN220 (ATCC67471) containing the origin of replication(ori) of the plasmid pBR322, for amplifiable replication in E. coli andthat of the broad host range plasmid pSa727 which permit replication inAgrobacterium (Tait, R. C. et al., Biotech 1, 269) (1982), as well asthe sequence comprising the cohesive end termini (COS) of thebacteriophage lambda, was digested with HindIII and treated with calfintestinal alkaline phaspatase. HindIII fragment No. 23 (H23) containingthe T-DNA right border and the nopaline synthase gene from theAgrobacterium plasmid Ti337, Bevan, M. et. al., supra, was isolatedfollowing HindIII digestion of MWB2341:H23 (obtained from W. Barnes).The H23 fragment was ligated to the HindIII-digested plasmid pSUN220 toproduce the plasmid pSUN221. The construction of pSUN221 is shown inFIG. 3.

4. Construction of pSUN473

The plasmid pSUN221 was digested with PstI and EcoRI and treated withmung bean nuclease. The plasmid pSUN470 was digested with PvuI andtreated with mung bean nuclease. The 3.1 kb PvuI fragment containing the35S promoter, APT II, 3'NOPS and B_(L) sequences was isolated andblunt-end ligated in 15% PEG to the treated pSUN221. The ampicillinresistance determinant of pSUN221 (Amp^(R)) was destroyed in theprocess. The resulting plasmid was identified as pSUN473. Theconstruction and partial map of pSUN473 are illustrated in FIG. 4.pSUN473, because of the pSa origin of replication, can be maintained inA. tumefaciens and is suitable for use as a binary vector for transferof genetically engineered T-DNA sequences to plants.

5. Construction of pSUN474

The plasmid pSV2-hph was obtained from C. Kado, University ofCalifornia, Davis, Calif., as a source of a gene which confersresistance to the antibiotic hygromycin (hph) useful as a transformationselection determinant in plants. pSV2-hph was digested with HindIII andBglII and a 1.4 kb fragment containing the hph gene was isolated andpurified.

The plasmid pBSM was obtained from Vector Cloning Systems, now known asStrategene Cloning System, La Jolla, Calif. and digested with HindIIIand BamHI followed by treatment with calf intestinal alkalinephosphatase. The 1.4 kb hph-fragment from pSV2-hph was then ligated tothe digested pBSM to produce pSUN474.

6. Construction of pSUN475

The plasmid pSUN474 was digested with HindIII and AvaI and the "stickyends" of the DNA fragments produced by this digestion were made blunt bytreatment with the Klenow fragment of E. coli DNA PolI in the presenceof dNTP's. A 1.3 kb fragment containing the hph gene was isolated andpurified.

The plasmid pSUN473 was digested with EcoRI, treated with the Klenowfragment of E. coli DNA PolI in the presence of dNTP's followed bytreatment with calf intestinal alkaline phosphatase. A fragment ofapproximately 13 kb was isolated and purified. This 13 kb fragment andthe 1.3 kb hph-fragment were blunt end ligated in the presence of 15%PEG to produce pSUN475. The filled-in EcoRI end of pSUN473 when ligatedto the filled-in AvaI end of the hph-fragment regenerated an EcoRI site.The construction and partial map of pSUN475 are illustrated in FIG. 5.

7. Construction of pSUN480

The plasmid pSUN214 (ATCC 67470) (see FIG. 1) was digested with BamHifollowed by treatment with the Klenow fragment of E. coli DNA PolI inthe presence of dNTP's to fill in the ends. Further digestion with EcoRIfollowed by treatment with calf intestinal alkaline phosphatasepermitted the isolation and purification of a 3.4 kb fragment containingthe 3'NOPS sequence and the pUC origin of replication and ampicillinresistance determinant sequences found in pSUN214.

The plasmid pSUN475 was digested with XbaI (which cuts in the multiplecloning site MCS) followed by treatment with the Klenow fragment of E.coli DNA PolI in the presence of dNTP's to fill in the ends. Theresulting linearized pSUN475 was then partially digested with EcoRI anda 215 kb fragment containing the CaMV 35S promoter and the hph codingsequence was isolated and purified. This fragment was ligated to the 3.4kb fragment from pSUN214 and the resulting plasmid was pSUN480 which isdepicted in FIG. 6

8. Construction of pSUN339 and pSUN340

The plasmid pYA208 (see FIG. 6) was digested with CfoI and treated withT₄ polymerase in the presence of dNTP's to generate blunt ends. Afragment of about 2280 base pairs containing an expressible lac fusionto the spaA sequence was isolated. Plasmid pYA208 contains a BamHIfragment containing the spaA gene in the correct orientation. The vectorpSUN480 was digested with PstI and EcoRI to remove the hygromycinresistance gene, and treated with T4 DNA polymerase and dNTP's togenerate blunt ends. The larger fragment of 4170 base pairs wasisolated. The lac-spaA fragment was ligated between the 35S and 3'NOPSsequences in the correct orientation in place of the hph-fragment toproduce the plasmid pSUN339. pSUN340 which has the lacZ-spaA insert inthe opposite orientation relative to the vector sequence was alsoisolated. (See FIG. 6)

9. Construction of pSUN341, pSUN342 and pSUN343

The plasmid pSUN339 was digested with ScaI and AsuII and the35S-lacZ-spaA-3'NOPS fragment of about 3263 base pairs was isolated. TheAsuII terminus of the fragment was filled in the dNTP's using the Klenowfragment of E. coli DNA polI.

The plasmid pSUN473 (FIG. 4) was digested with XbaI, which has a singlesite in the multiple cloning site sequence and the 5'end was filled inwith dNTP's using the Klenow fragment of E. coli DNA polymerase I. The35S-lacz-spaA-3'NOPS expression cassette from pSUN339 was ligated to thedigested plasmid pSUN473 in different orientations with regard to the35S-APTII-3'NOPS sequence of the vector. The plasmid pSUN341 containsthese sets of sequences in a head to head orientation. The plasmidpSUN343 contains these sets of sequences in a head to tail orientation.Plasmid pSUN342 was constructed in a similar fashion using pSUN340 asthe starting material for ScaI-AsuII digestion. The plasmids pSUN344 andpSUN345 are independent isolates identical to pSUN343. The plasmidpSUN346 is an independent isolate identical to pSUN341. The constructionof pSUN341, pSUN342 and pSUN343 are illustrated in FIG. 6. pSUN341 andpSUN343 have opposite orientations of the insert relative to the vectorbut with the CaMV 35S and lac promoters in the same correct orientationsto permit SpaA expression in both E. coli and plants. pSUN341 in E. coliDH5α and pSUN343 in E. coli DH5α were deposited at the ATCC under theBudapest Treaty on Aug. 31, 1988 and assigned the numbers 67,787 and67,785, respectively. pSUN342 was constructed as a control using theScaI to AsuII fragment of pSUN340 into XbaI cut pSUN473. In thisconstruct SpaA should be synthesized in E. coli under the control of thelac promoter but not in plants since the CaMV 355 promoter is in thewrong orientation (see FIG. 6).

B. Construction of pSUN387

The plasmid pSUN387 contains components of the plasmids pUC18, pSUN335and pSUN491 (ATCC No. 67786) is deposited under the Budapest Treaty. ThepUC component includes all sequences outside of the EcoRI and HindIIsites of the multiple cloning region. This contains the origin ofreplication and the gene for ampicillin resistance. The sequences frompSUN491 include the CaMV 35S promoter with a tandem duplication of a 327bp HincII to EcoRV fragment which contains sequences shown to confertranscriptional enhancement to the 35S promoter and other heterologouspromoters in plant systems. Kay et al., Science 236, 1299 (1987). Also,a multiple cloning region containing sites for NcoI, BamHI, XbaI, SalI,PstI and EcoRI followed by about 681 base pairs of 3'NOPS sequence areincluded downstream of the 35S promoter. The plasmid pSUN335 providestwo sequences to permit the expression of genes in bacteria wheninserted between the 35S promoter and 3'NOPS. These include a synthetic17 bp KpnI to NcoI fragment which contains a perfect Shine--Dalgarnosequence, optimally spaced from the ATG contained within the NcoI siterequired for initiation of protein synthesis. Shine and Dalgarno, Proc.Natl. Acad. Sci. USA 71, 1342 (1974). Also included, upstream of the 35Spromoter, is the promoter from the asd gene of Streptoccus mutans whichhas been shown to be a strong promoter of transcription in E. coli.Cardineau and Curtiss, J. Biol. Chem. 262, 3344 (1987).

A map of pSUN387 is seen in FIG. 7. The sequence of the plasmid is alsoprovided in FIG. 8. pSUN387 is deposited under the Budapest Treaty andis assigned ATCC No. 31985.

C. Construction of pSUN390, pSUN391, pSUN392, pSUN393 and pSUN394

The plasmids pYA177, pYA178, pYA179 and pYA180 (Curtiss et al, Vaccine,1988, supra) possess 1, 2, 3 or 4 copies of a 483 base pair SstI t SstIfragment respectively, specifying the major antigenic/immunogenicdeterminant of the SpaA protein followed by a C terminalantigenic/immunogenic determinant of the SpaA protein specified byapproximately 1204 base pairs. Cloning was accomplished by digestingpSUN387 with XbaI which cuts the multiple cloning site, generating bluntends using the Klenow fragment of DNA polymerase 1 and then digestedwith NcoI. The fragments specifying SpaA determinants were cut out ofpYA177, pYA178, pYA179 and pYA180 by first digesting with HindIII,treating with Klenow and then cutting with NcoI after which thefragments were ligated into the prepared pSUN387 DNA.

As revealed by analysis of the complete nucleotide sequence of pSUN387(see FIG. 8), expression of SpaA in E. coli is under the control of theS. mutans asd promoter and in plants under the control of the CaMV 35Spromoter. In this construct, there are no ATG start codons following theCaMV 35S promoter prior to the ATG start codon at the NcoI site whichinitiates the reading frame for all of the SpaA inserts in pSUN390,pSUN391, pSUN392, pSUN393 and pSUN394. E. coli HB101 (pYA726) with aSpaA insert is deposited under ATCC No. 31985.

II. Expression and Stability of SpaA Protein

FIG. 9 shows a Western blot analysis of transformed E. coli DH5αexpressing the SpaA protein due to the presence of pSUN341, pSUN342,pSUN343, pSUN344, pSUN345 and pSUN346. SpaA occurs at about 116 kDa.Expression of SpaA in E. coli is independent of the orientation of theCamV promoter but is dependent on the correct orientation of the lacpromoter. SpaA breakdown products occur primarily in the region fromabout 60 kDa to about 115 kDa. It is apparent from this analysis thatantibody against SpaA recognizes all forms of the protein, native aswell as breakdown products. This is advantageous since breakdownproducts could occur in planta as well as in the intestine.

FIG. 10 shows a Western blot analysis of SpaA protein synthesis by E.coli containing recombinant plasmids specifying SpaAantigenic/immunogenic determinants. The recombinant plasmidspYA177pYA180 are contained in E. coli _(x) 2991 whereas all of thepSUN390- pSUN394 recombinant vectors are contained in E. coli DH5α.pSUN390 and pSUN391 each specify one of two major bands specified bypYA177. The reason for this is not known. It is apparent that pSUN393 isnot behaving in an expected way with regard to production of SpaA. Uponinitial isolation it caused a much higher level of SpaA synthesis. Allof the pSUN plasmid constructs cause synthesis of less SpaA than the pYAconstructs. This is most likely because the S. mutans asd promoter issome 1250 base pairs away from the ATG start codon for SpaA synthesis inthe pSUN vectors whereas the distance between the trc promoter and theATG start in the pYA vectors is only 45 base pairs. The SpaA pYA180 hadmolecular masses of 94, 116, 145 and 164 kDa, respectively. Again SpaAbreakdown occurs in E. coli but these breakdown products are recognizedby the antibodies against the SpaA native protein.

Prior to conducting studies to see whether E. coli expressing SpaA couldelicit an immune response after oral feeding to mice, studies wereundertaken to investigate the stability of SpaA protein expressed in E.coli to various food processing regimens. In general, heating of E. coli_(x) 2846 possessing pYA210 (a recombinant vector similar to pYA208depicted in FIG. 6 but containing three tandem repeats, all in the samereading frame, of the 2.0 kb BamHI fragment in pYA208 specifying SpaA)to temperature of 80° C. or above for 10 minutes or more completelystabilized SpaA from breakdown during storage at room temperature or inthe cold. Attempts to examine the stability of purified SpaA protein orof SpaA protein released by lysed cells of E. coli _(x) 2846 containingpYA210 when mixed with mouse meal were hampered by the fact thatconstituents in mouse meal interfered with SDS gel electrophoresis andWestern blot analysis. It was thus not possible to accurately quantitatestability of SpaA antigenicity in mouse meal either before or afteringestion. Nevertheless, immunogenicity is an excellent indicator ofstability during food processing and digestion since the antigen mustsurvive to arrive in the small intestine to be taken up by M cellsoverlying the gut-associated lymphoid tissue.

III. Immunogenicity of SpaA Protein

Plant material containing heat-killed and lysed E. coli _(x) 2846/pYA210was lyophilized and ground to a meal. It was then stored in the drystate. Based upon protein, as described above, mouse meal was preparedso as to have 25 to 500 nanograms of SpaA protein per gram of meal. Thisdiet was fed ad libitum to female BALB/c mice, 9 to 10 weeks old. Themice were weighed weekly to follow their growth and development. Micewere observed visually to determine the status of their health.

Saliva samples were collected weekly. Salivation was stimulated bypilocarpine. Serum was collected biweekly using retroorbital bleeding.

Serum anit-SpaA IgG and salivary IgA were detected by ELISA. dynatechLaboratories immulon-1 flat-bottom polystyrene plates were coatedovernight at 41° C. with 100 μl (4.25 μg protein) of a 1:5 dilution (in0.1M NaHCO₃ buffer, pH 9.6) of semi-purified SpaA (obtained from a 70%ammonium sulfate precipitated filtered supernatant fluid from S. mutansfollowed by dialysis and lyophilization) or of SpaA purified fromrecombinant E. coli. Plates were then washed three times with phosphatebuffered saline (PBS; pH 7.2) containing 0.05% Tween-20 and then blockedfor 90 min. with PBS plus 0.05% Tween-20 and 1% bovine serum albumin.After washing, serum samples (100 μl of each dilution) were added andallowed to incubate overnight at 4° C. Plates were washed again and thesecondary antibodies which were affinity purified goat anti-mouse IgG(chain specific) or goat anti-mouse IgA (γ-chain specific) conjugatedwith alkaline phosphatase (1:1000 dilution) added and incubated for 4hours at room temperature. After washing, nitrophenyl phosphatesubstrate dissolved in diethylalamine buffer, pH 9.8, was added andplates incubated at room temperature for 1.5 hours. They were then readat 405 num, with a Bio-Tek Automated EIA Plate Reader. Standardizationof anti-SpaA serum IgG and serum IgA in comparison to total serum IgAand IgG were accomplished by use of purified mouse IgG myeloma proteinas a standard in ELISA or the purified IgA myeloma protein.

Salivary anti-SpaA IgA were quantified in analogous manners usingaffinity purified rabbit anti-mouse (α-chain specific) alkalinephosphatase conjugate as the secondary antibody. Since pilocarpinestimulation causes variable dilution of saliva, it was essential toquantitate the specific amount of anti-SpaA sIgA in saliva in comparisonto total sIgA, the later determined by using the mouse myeloma IgA as astandard. Suitable positive and negative controls were used. For serumantibody, mouse sera obtained from mice immunized with purified SpaAprotein obtained from recombinant E. coli were used. For positivecontrols for salivary secretory IgA, mice were immunized directly in thesalivary glands, an immunization route known to induce high levels ofsIgA specific against the immunizing antigen. It should be noted thatthe measurement of antibody titers in saliva make use of SpaA proteinpurified from recombinant E. coli. This is because the conventional miceused have antibodies against common streptococcal antigens, includinglipoteichoic acid, and these contaminating antigens are difficult toseparate from SpaA protein obtained from supernatant fluids of S. mutanscultures.

Table 1 shows the results of experiments on long term feeding ofmicroorganisms expressing the SpaA protein to mice. Table 1 shows sIgAtiters in saliva of mice fed E. coli which express the SpaA protein.

                  TABLE 1                                                         ______________________________________                                        sIgA titers in saliva of BALB/c mice fed E. coli χ2846                    (pYA210) expressing SpaA protein.sup.a                                        Control                    χ2846                                                                             (pYA210)                                                 Anti-    Percent                                                                             Fed   Anti- Percent                              Time   Total  SpaA-    spec. Total SpaA- spec.                                (weeks)                                                                              sIgA.sup.b                                                                           sIgA.sup.b                                                                             sIgA  sIgA.sup.b                                                                          sIgA.sup.b                                                                          sIgA                                 ______________________________________                                        0      2023   2.6      0.13  3852  4.1   0.11                                 2      4913   3.4      0.17  4785  4.3   0.09                                 4      4249   3.4      0.08  5969  6.9   0.12                                 7      5400   3.9      0.07  5351  5.0   0.09                                 9      7046   4.9      0.07  5455  49.1  0.90                                 11     6994   6.8      0.10  6502  77.4  1.19                                 13     6428   17.8     0.28  4772  58.1  1.22                                 16     6351   4.5      0.07  8089  85.7  1.06                                 18     6825   5.5      0.08  9526  40.3  0.42                                 21     7891   7.1      0.09  7512  61.0  0.81                                 26.5   5846   4.2      0.07  6728  45.5  0.68                                 28.5   5904   3.1      0.05  7232  53.8  0.74                                 ______________________________________                                         .sup.a E. coli χ2846 (pYA210) was heat killed, lyophilized and added      to mouse meal at a concentration equivalent to 10.sup.7 bacteria per gram     of mouse meal which was fed ad libitum. Saliva samples were collected         following pilocarpine injection. Total sIgA and antiSpaA sIgA were            quantitated by ELISA.                                                         .sup.b Expressed in ng/ml of saliva.                                     

Example 2

Agrobacterium tumefaciens--mediated transformation

I. Vector Construction excised from these vectors and introduced into abinary vector such as pSUN473 (FIG. 4) prior to transfer to A.tumefaciens. In each case the binary vector such as pSUN341 would betransferred to an A. tumefaciens strain possessing a disarmed Ti plasmidby triparental mating, Fraley et al., supra. This could be accomplishedby use of an A. tumefaciens strains such as LBA4404 or LBA1050possessing disarmed plasmids such as pAL4404 or pAL1050. A. tumefaciensstrains containing pSUN341 and pSUN343 produced as much SpaA protein asdid E. coli strains with these vectors as revealed by Western blotanalysis (data not shown).

II. Transformation

Nicotiana tobaccum, varieties havana and Xanthi, have been transformedby A. tumefaciens containing pSUN341 and pAL4404 or pSUN343 and pAL4404using the leaf disc transformation method (Horsch et al., supra).Briefly, axenic leaf tissues prepared as discs were dipped in a liquidculture of A. tumefaciens at a concentration of ˜10⁸ cells/ml. Afterallowing sufficient time for the infection to occur (5-30 sec.) thetissues were blotted dry and plated on tissue regeneration medium. After2 or 3 days, the explant tissues were removed to fresh medium containingthe antibiotics carbenicillin or defotaxime to kill the A. tumefaciensand kanamycin to select for transformed plant cells. In tobacco, it isfairly easy to generate shoots which can form whole transgenic plants.The transformed tobacco tissue was selected and whole plants regeneratedin accordance with the procedures described by Rogers et al., MethodsEnzymol. 118, 627 (1986). Callus tissue was assayed for nopalinesynthase activity in accordance with Otten et al., Biochem. biophys.Acta. 527, 497 (1978). A total of 64 transgenic plants, derived from 5separate experiments regenerated from callus tissue growing on selectedmedia with 300 μug kanamycin/ml, were tested for the production of SpaAprotein using dot blot and Western blot analyses and for production ofnopaline using paper electrophoresis with a nopaline standard and anegative control plant. Only one of 64 plants produced SpaA whereas 33of 46 tested produced nopaline. DNA was isolated from a number of plantsfor analysis using the Southern blot technique, Southern, J. Mol. Biol.98, 503 (1978). By using a 2.0 kb SpaA probe, six plants tested weredemonstrated to contain the SpaA gene regardless of whether they testednegative or positive for nopaline production or SpaA synthesis. Using aDNA probe for neomycin phosphotransferase to analyses restricted DNAfrom nine plants revealed that they all contained the neomycinphosphotransferase gene and all had DNA insertions in different regionsof the tobacco genome since the flaking sequences were different in allnine instances. The one plant making SpaA protein was nopaline positiveand contains the SpaA gene sequence from pSUN343.

III. Production and Stability of SpaA in Transgenic Tobacco

SpaA protein produced by E. coli containing pYA177 was purified bypreviously developed methods (Holt et al., supra) and followingsuccessive separation on SDS polyacrylamide gels by electroelution ofthe highest molecular weight SpaA band. Leaf discs from the transgenictobacco plant producing SpaA protein were homogenized (using a WheatonInstruments overhead stirred containing a microfuge pestle) in 20 mmTris pH 7.4, 350 mM Nacl and 0.1% β-mercaptoethanol. The supernatantfluid was recovered after centrifugal sedimentation of debris. Proteinassays were done on the purified SpaA protein and on the tobacco cellextract. Various dilutions of the Tobacco cell extract and varyingamounts of purified SpaA protein in various lanes as controls wereelectrophoresed on SDS polyacrylamide gel. The gel was then subjected towestern blot analysis using rabbit anti SpaA serum. The results of thisanalysis are depicted in FIG. 11. The SpaA protein produced by thetransgenic tobacco has a mass of 105 kDa which is slightly less than thesize of the SpaA protein made by pYA208 and pSUN343 (FIG. 6). Thedifference in size of the protein is probably due to processing inplanta. The SpaA protein produced by the transgenic plant is a doubletand there is little or no breakdown material discernible. This is not tosay that breakdown does to occur but that if it does it is degraded bythe plant. The intensities of the Western blot bands were quantitatedusing a Molecular Dynamics densitometer. The data used to derive astandard curve are included in Table 2. Based on this, it was calculatedthat the SpaA protein synthesized by the transgenic tobacco plantrepresented 0.2% of the total plant protein.

                  TABLE 2                                                         ______________________________________                                        Densitometer readings of the bands shown on                                   the Western blot depicted in FIG. 11.                                         Purified              Tobacco                                                 SpaA    Densitometer  extract  Densitometer                                   (ng)    value         (μg)  value                                          ______________________________________                                        10       145466        25       75423                                         25       267154        50      169168                                         50       1696040       75      219917                                         75      1044156       100      257764                                         100     1205834       150      430530                                         150     1534383       200      519636                                         ______________________________________                                    

IV. Heritability of the Ability to Produce the SpaA Protein.

The SpaA producing tobacco plant was permitted to form seed. After seedcollection and curing, 50 seedlings representing the F2 generation wereobtained after seed germination and plants grown to test forheritability of the SpaA encoding sequence. Dot blot and Western blotanalyses were used to detect and quantitate SpaA production. Eighteenplants did not make any SpaA protein whereas 32 did. These all produceda SpaA protein having the same molecular mass as the SpaA proteinproduced by the parental transgenic tobacco plants. The _(x) ² value is3.23 which falls below the _(x) ² value for probability of 0.05 whichdemonstrates that the 32:18 ratio of SpaA producing to non-producingplants fits the expected 3:1 ratio of a plant heterozygous for a traitthat is segregating as a single Mendelian factor.

The 32 SpaA positive plants were further analyzed by quantitativedesitometric measurement of Western blot data to determine whetherplants homozygous for the ability to produce SpaA could bedifferentiated from plants that were heterozygous for the trait. Thedata in Table 3 reveal that twelve of the plants produced an amound ofSpaA that might be indicative of homozygosity. Six of these plants aswell as six judged to be heterozygous are being grown for production ofseed to determine, by analysis of germinated offspring, whether thedensitometric quantitation can be relied on to indicate homozygosityversus heterozygosity.

The stability and immunogenicity analyses are performed as describedabove in Example 1.

                  TABLE 3                                                         ______________________________________                                        SpaA protein as a percentage of total protein                                 in 32 F2 SpaA producing plants.*                                                      SpaA × 100       SpaA × 100                               Plant # total protein  Plant # total protein                                  ______________________________________                                         2      .043           25      .008                                            4      .049           26      .016                                            6      .038           27      .026                                            9      .032           30      .049                                           11      .035           31      .023                                           13      .044           33      .013                                           14      .008           34      .011                                           15      .015           35      .014                                           17      .022           37      .026                                           18      .012           38      .009                                           19      .002           39      .015                                           20      .031           42      .014                                           21      .038           43      .009                                           22      .003           48      .003                                           23      .015           49      .011                                           24      .020           50      .007                                           ______________________________________                                         *Values were calculated from laser densitometer readings of a Western         blot. Plants 1, 3, 5, 7, 8, 10, 12, 16, 28, 29, 32, 36, 40, 41, 44, 45, 4     and 47 did not produce any SpaA protein.                                 

Processing of Transgenic Plant Material for Use as Animal Feed.

Leaf tissue from a SpaA producing plant was removed, cut into small(approximately 3 cm²) pieces and dried at 37° C. for approximately 2days. Also, leaf tissue from a SpaA producing plant was removed, quickfrozen in liquid nitrogen and lyophilized using a Vertis freeze mobileII lyophilizer. In Western blot analysis the amount of SpaA protein as apercentage of total protein, from both methods, revealed little or noloss of SpaA protein. Lyophilized leaf tissue from the transgenictobacco plant producing SpaA was stored at -20° C. and at roomtemperature for 13 days. Lyophilized tissue stored at room temperaturefor 13 days was also mixed with mouse meal at a 1:1 ratio. All sampleswere homogenized using a Wheaton instruments overhead stirrer containinga microfuge pestle in 20 mM Tris pH 7.4, 350 mM NacL and 0.1%mercaptoethanol. The supernatant fluid was recovered after centrifugalsedimentation of debris followed by protein assay. Western blot analysisshowed little or no loss of SpaA protein in all cases (FIG. 12). Plantsamples in lanes 2, 4, 6 and 8 were from a transformed plant thatcontains the spaA sequence but which did not express the SpaA protein asrevealed by the absence of reaction with spaA antiserum. See lanes 2 and4. However, when stored for 13 days at room temperature something in thelyophilized plant tissue reacts weakly with the SpaA antibody as seen inlanes 6 and 8. This is not completely understood.

Immunogenicity of SpaA Protein Expressed in Plants. SpaA producingtobacco plant material processed as described above can be mixed withmouse meal at different dosages to investigate elicitation of asecretory immune response against the ingested SpaA protein. Previousresults by Michalek et al., 1976, supra, observed significant sIgAproduction in rats give 10⁸ killed S. mutans cells/ml of drinking water.The SpaA protein in S. mutans represents approximately 0.2% of the totalprotein and each S. mutans cell contains approximately 2×10⁻¹⁰ mg ofprotein. Thus, there are 40 nanograms of SpaA in every 10⁸ S. mutanscells. Based on the foregoing analysis of the heterozygous SpaAproducing transgenic plant, 2 mg of dried transgenic tobacco contains 40nanograms of SpaA protein. Mouse meal can be supplemented with 200 μg, 2mg, and 20 mg of dried transgenic tobacco meal per gram of mouse meal.These concentrations provide oral immunization doses comparable toadministering 10⁷, 10⁸ and 10⁹ S. mutans cells per gram of feed. Forthis experimental protocol, it should be noted that maximal doses oftobacco meal will constitute 2% of the mouse meal diet. 2% is 5 timesless than the dosage of tobacco meal that can be tolerated by continuousconsumption by mice without any noticeable adverse physiologicaleffects.

V. Immunogenicity of SpaA Protein Expressed in Plants

This diet is fed ad libitum to female BALB/c mice, 9 to 10 weeks old.The mice are weighed weekly to follow their growth and development. Miceare observed visually to determine the status of their health.

Saliva samples are collected weekly. Salivation is stimulated bypilocarpine. Serum is collected biweekly using retroorbital bleeding.

Serum anti-SpaA IgG and salivary IgA are detected by ELISA. DynatechLaboratories immulon-b 1 flat bottom polystyrene plates are coatedovernight at 41° C. with 100 μl (4.25 μg protein) of a 1:5 dilution in0.1M NaHCO₃ buffer, pH 9.6, of semi-purified SpaA, obtained from a 70%ammonium sulfate precipitated filtered supernatant fluid from S. mutansfollowed by dialysis and lyophilization, or of SpaA purified fromrecombinant E. coli. Plates are then washed three times with phosphatebuffered saline (PBS; pH 7.2) containing 0.05% Tween-20 and then blockedfor 90 min. with PBS plus 0.05% Tween-20 and 1% bovine serum albumin.After washing, serum samples (100 μl of each dilution) are added andallowed to incubate overnight at 4° C. Plates are washed again and thesecondary antibodies which are affinity purified goat anti-mouse IgG(γ-chain specific) or goat anti-mouse IgA (α-chain specific) conjugatedwith alkaline phosphatase (1:1000 dilution) added and incubated for 4hours at room temperature. After washing, nitrophenyl phosphatesubstrate dissolved in diethylalamine buffer, pH 9.8, is added andplates incubated at room temperature for 1.5 hours. They are then readat 405 nm, with a Bio-Tek Automated EIA Plate Reader. Standardization ofanti-SpaA serum IgG and serum IgA in comparison to total serum IgA andIgG are accomplished by use of purified mouse IgG myeloma protein as astandard in ELISA or the purified IgA myeloma protein.

Salivary anti-SpaA IgA are quantified in analogous manners usingaffinity purified rabbit anti-mouse (α-chain specific) alkalinephosphatase conjugate as the secondary antibody. Since pilocarpinestimulation causes variable dilution of saliva, it is essential toquantitate the specific amount of anti-SpaA sIgA in saliva in comparisonto total sIgA, the later determined by using the mouse myeloma IgA asstandard. Suitable positive and negative controls are used. For serumantibody, mouse sera obtained from mice immunized with purified SpaAprotein obtained from recombinant E. coli are used.

Example 3 Plant Transformation

I. Construction of Vectors

Vectors pSUN343 described in Example 1 containing the SpaA sequence isused.

II. Plant Transformation by Electroporation

The procedure used to electroporate tobacco protoplasts is essentiallyas described by David Cheng and co-workers in the Hoefer ScientifcInstruments Technical Bulletin #118. The upper epidermis of tobaccoleaves (Nicotiana tobacum c.v. Havana) isolated when 3 or 4 cm in lengthfrom in vitro grown plants, is brushed with 320 grit aluminum oxidepowder to permit the infiltration of cell wall degradative enzymes usedto prepare protoplasts by the method of Magnien, E. et al., ActaGenetica Sinica 7, 231 (1980). Enzymatically released protoplasts arewashed with 17.5% sucrose, floated and harvested by centrifugation for 5min. at 300×g in a 60 ml Babcock bottle. Linearized or supercoiled DNA(pSUN343) is mixed with the protoplasts in a final volume of 0.5 ml at aconcentration of 0.1 mg/ml and 7×10⁵ cells/ml respectively, in a 16 mmdiameter Nunc Multidish well. A single pulse is administered at roomtemperature (23° C.) with a Hoefer PG 101 Progenetor electroporationunit using a PG120-2.5 electrode for 10 msec at 200 V. Electroporatedprotoplasts are kept stationary for 10 min. prior to the addition of 1ml of culture medium. Cells were subsequently diluted to a finalconcentration of 10⁵ cells/ml. These cells may then be assayed fortransient expression of the spaA gene after a period of 40-48 hours or,depending on the DNA construct used, plated to generate callus tissuesunder kanamycin selection, followed by regeneration to whole plants.

III. Regeneration

Post-transformation protoplasts are plated on callus proliferationmedium with kanamycin as selection pressure and cultured for 2-3 weeksat 24° C. in a 16 hour diffused light/8 hour dark cycle. Callus issubcultured every 2-3 weeks to produce enough tissue to proceed withregeneration. After enough tissue is obtained, the callus is transferredto regeneration medium with or without selection pressure and culturedfor 3-4weeks at 24° C. in a 16 hour diffused light/8 hour dark cycleuntil shoot bud formation. At this time, the material is transferred toplant establishment medium with or without selection pressure andcultured at 24° C. in a 16 hour diffused light/8 hour dark cycle until3-4 leaves formed. The plantlet is then transferred to soil.

The callus tissue and regenerated plants can be evaluated for level ofSpaA protein, relative to total protein by ELISA or by Western blot andquantitative densitometer analysis. (See Tables 2 and 3).

IV. Stability, Heritability and Immunogenicity of SpaA Protein Expressedin Plants

Stability, heritability and immunogenicity of SpaA protein intransformed plants are analyzed by the method of Examples 1 and 2.

Example 4

Example 2 is repeated except that in step I a suitable planttransformation vector containing the gtfB gene is constructed. Forexample, pSUN387 (gtfB) is prepared which contain the gtfB gene isolatedfrom pSU20 (shiroza, T. et al., supra) in place of the spaA gene. ThegtfB encoding sequence along with the CaMV 355 promoter and NOPS 3'polyA sequence is introduced into an appropriate binary vector such aspSUN473 or pSUN475.

Following generation of transgenic tobacco plant, stability,heritability and immunogenicity analyses for GtfB protein are performedas described in Examples 1 and 2.

Example 5

Example 3 is repeated except that in step I a vector containing bothspaA and gtfB is constructed. For example, gtfB is inserted to followthe spaA sequence in pSUN394. In this way a construct expressing twocolonization antigens is formed.

Following generation of transgenic plants from protoplasts-derivedcallus, stability, heritability and immunogenicity of SpaA and GtfBproteins are analyzed as described in Examples 1 and 2.

Example 6

Example 3 is repeated except that in step I a suitable planttransformation vector containing the dextranase gene is constructed. Forexample, pSUN387 is prepared which contains the dextranase (dex) geneisolated from pYA993.

Following generation of transgenic plants from protoplasts-derivedcallus, stability, heritability and immunogenicity of dextranase proteinare analyzed as described in Examples 1 and 2.

Example 7

Example 3 in repeated except that in step I a suitable planttransformation vector containing both spaA and dex is constructed. Forexample, dex is inserted to follow the spaA sequence in pSUN394. In thisway a construct expressing two colonization antigens is formed.

Following generation of transgenic plants from protoplasts-derivedcallus material, stability, heritability and immunogenicity of SpaA anddextranase proteins are analyzed as described in Examples 1 and 2.

Example 8

Example 3 is repeated except that in step I a suitable planttransformation vector containing the K88 pilus colonization antigen geneis constructed. For example, pSUN387 is prepared which contains the K88pilus colonization antigen isolated from plasmid pMK005, which wasdeveloped by Kehoe et al., Nature 291, 122 (1981). Following generationof transgenic plants from protoplasts-derived callus material,stability, heritability and immunogenicity analyses of K88 antigen areperformed as described in Examples 1 and 2.

Example 9

Example 3 is repeated except that in step I a suitable planttransformation vector containing the K99 pilus colonization antigen geneis constructed. For example pSUN38 is prepared which contains the K99pilus colonizations antigen gene isolated from plasmid pRI9906.Following generation of transgenic plants from protoplasts-derivedcallus material, stability, heritability and immunogenicity analyses ofK99 antigen are performed as described in Examples 1 and 2.

Example 10

Example 3 is repeated except that the plant transformation vector is theplasmid pSUN387 (spaA/LT-B) containing a DNA sequence which codes for afusion protein comprising the SpaA protein and the LT-B protein. TheLT-B sequence is the N-terminus of the fusion protein. A DNA sequencecoding for the LT-B protein is isolated from E. coli (Yamaoto, T andYokoto, T., supra). Following generation of transgenic plants fromprotoplasts-derived callus material, stability, heritability andimmunogenicity analyses of SpaA and LT-B proteins are performed asdescribed in Examples 1 and 2.

Example 11

Example 2 is repeated except that plant transformation using pSUN473(gtfB) was carried out on tomato according to Fillatti, J. et. al,(1987), supra. Following generation of whole plants from selectedexplant tissues, stability, heritability and immunogenicity analyses ofgtfB are performed as described in Examples 1 and 2.

Example 12

Example 2 is repeated except that plant transformation using pSUN475(LT-B) was carried out on sunflower according to Everett, N. P. et al.,(1987), supra. Following generation of whole plants from selectedexplant tissues, stability, heritability and immunogenicity analyses ofLT-B protein are performed as described in Examples 1 and 2.

Example 13

Example 2 is repeated except that plant transformation using pSUN473(K99) was carried out on soybean according to Hinchee, M. A. et al.,(1987), supra. Following generation of whole plants from selectedexplant tissues, stability, heritability and immunogenicity analyses ofK99 protein are performed as described in Examples 1 and 2.

Example 14

Example 2 is repeated except that plant transformation using pSUN473(K88) was carried out on potato according to Faciotti, D. et al.,(1985), supra. Following generation of whole plants from selectedexplant tissues, stability, heritability and immunogenicity analyses ofK88 antigen are performed as described in Examples 1 and 2.

Example 15

Plant transformation is carried out by microinjection on alfalfa.

Transfer of pSUN387 (K99) into plant cells is achieved by injection of asolution of plasmid DNA with a finely pulled glass needle directly intoisolated protoplasts, cultured cells and tissues as described Reich, T.J. et al., Bio/Technology 4, 1001, (1986); Can.J.Bot. 64, 1259, (1986)and injection into meristematic tissues of seedlings and plants asdescribed by De La Pena, A. et al., Nature 325, 274, (1987), Graves, A.C. et al., Plant Mol. Biol. 7, 763, (1984).

Stability, heritability and immunogenicity analyses of K99 protein areperformed as described in Examples 1 and 2.

Example 16

Plant transformation is carried out by application of polyethyleneglycol on tobacco according to Negrutiu, R. et al., (1987), supra. TheDNA used is linearized plasmid pSUN390.

The protoplasts are suspended in 0.5 M mannitol containing 15 mM MgCl₂at a density of about 2×10⁶ per ml. the protoplasts suspension isdistributed into 10 ml plastic centrifuge tubes. The DNA is added andthen the PEG solution added 40% (w/v MW 4000 in 0.4M mannitol, 0.1MCa(NO₃)₂, (pH 7.0)!. The solution are mixed gently and incubated for 30minutes at room temperature (about 24° C.) for 30 minutes withoccasional shaking. Wash solution is then added, and the contents of thetube gently mixed. The wash solution consists of 87 nM mannitol, CaCl₂,MgCl₂, KCl, Tris/HCl and m-inositol, (pH 9.0). Four further aliquots ofwash solution are added at 4 minute intervals, with mixing after eachaddition. The tube is then centrifuged at about 60 g for about 10minutes, and the supernatant discarded. The sedimented protoplasts aretaken up in culture medium, and placed in a 10 cm petri dish.

Stability, heritability and immunogenicity analyses of SpaA protein areperformed as described in Examples 1 and 2.

Example 17

Example 16 is repeated except that in step II plant transformationpSUN387 (K88) is carried on Lolium multiflorum according to Negrutiu, R.et. al., (1987), supra. Stability, heritability and immunogenicityanalyses of K88 protein are performed as described in Examples 1 and 2.

Example 18

Transformation of Rice by Electroporation

DNA transfer and selection of transformants. Protoplasts are isolatedfrom anther-derived cell suspensions or ice (Oryza sativa), andelectroporated according to Fromm et al., with some modification, asfollows. Protoplasts (2×10⁵) and circular-form plasmid such as pSUN390,pSUN391, pSUN392, pSUN393 and pSUN394 (10 μg each) are suspended in 0.6ml of a buffer consisting of 0.5 mM 2- N-Morpholino!ethanesulfonic acid(pH 5.8), 7 mM KCl, 4 mM CaCl₂ --2H₂ O and 6.5% mannitol in a plasticcurvet (inter-electrode distance was 0.4 cm). An electrical pulse isdelivered from a 125 μF capacitor charged at 500 V/cm (Gene-Pulser,Bio-Rad, Calif., USA). The resistance-capacitance (RC) time-constantsare 4 msec and 20 msec, respectively. After 10 min at 4° C., followed by10 min at room temperature, electroporated protoplasts are transferredto a petri-dish (5 cm in diameter) containing 2.5 ml B5 mediumsupplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 5%mannitol. After 2 weeks, 1 ml NO₃ medium (B5 medium without ammoniumsulphate) supplemented with 2 mg/l 2,4-D and 3% glucose is added. After3 weeks, the medium is replaced by NO₃ medium lacking glucose, andcontaining 2 μg/ml G418 sulphate (Schering Co., N.J.). One month afterelectroporation, serving microalli are transferred to NO₃ mediumcontaining 20 μg G418/ml and 1% agarose (Sigma type I). After another 2weeks, growing calli are transferred onto N6 medium containing 0.2 mg/lindole-3-acetic acid, 1 mg/l kinetin and 1% agarose (regenerationmedium). Callus tissue is assayed for nopaline synthase activity inaccordance with Otten et al., supra.

Stability, heritability and immunogenicity analyses of SpaA protein areperformed as described in Examples 1 and 2.

Example 19

Stable Transformation of Soybean by Particle Acceleration

Another method to introduce foreign DNA sequences into plant cellscomprises the attachment of said DNA to tungsten particles which arethen forced into plant cells by means of a shooting device as describedby Klein, T. M. et al., supra or by means of particle acceleration usinga finely tuned electric discharge to accelerate DNA coated goldparticles as described by McCabe, E. T. et al., supra. Any plant tissueor plant organ may be used as the target for this procedure, includingbut not limited to embryos, apical and other meristems, buds, somaticand sexual-tissues in vivo and in vitro. Transgenic cells and callus areselected following established procedures known in the art. Targetedtissues are induced to form somatic embryos or regenerate shoots to givetransgenic plants according to established procedures known in the art.The appropriate procedure may be chosen in accordance with the plantspecies used.

The regenerated plant may be chimeric with respect to the incorporatedforeign DNA. If the cells containing the foreign DNA develop into eithermicro-/or macrospores, the integrated foreign DNA will be transmitted tosexual progeny. If the cells containing the foreign DNA are somaticcells of the plant, non-chimeric transgenic plants are produced byconventional methods of vegetative propagation either in vivo, i.e. frombuds or stem cuttings, or in vitro following established proceduresknown in the art. Such procedures may be chosen in accordance with theplant species used.

Transformation is carried out on soybean according to McCabe, D. E. etal., (1988), supra.

DNA preparation. DNA coated projectiles are prepared by mixing 1.5-3 μmgold spheres (Alfa Chemical Co.) with a solution of pSUN387 (gtfB) DNAat a rate of 1 mg gold beads per 1 μg of DNA. The slurry is dried undera stream of N₂, and the dry pellet resuspended in 100% ethanol at aconcentration of 2 mg beads per ml. 162 μl of the gold suspension ispipetted onto an 18 mm square of aluminzed plastic film. The sheet, nowcarrying a thin layer of beads, is air dried.

Particle acceleration. Embryonic axes with their primary leaves removedto expose the meristem, are the beads is loaded onto a particleaccelerating machine, which uses the discharge of a high voltagecapacitor through a small water droplet as the motive force. A 100-meshretaining screen is placed between the sheet and the target tissuesuspended above the machine. The assembly is then evacuated to about 500mm Hg to reduce aerodynamic drag. Fourteen kV from a 2 μF capacitor isdischarged through a 10 μl water drop inside the polyvinyl chlorideexpansion chamber. The sheet is blown against the retaining screenpermitting the beads to continue onward to impact the target tissuessuspended above the screen. The target axes are positioned on a wateragar plant so that, when the plate is inverted over the screen, themeristematic regions are positioned in the path of the acceleratedbeads.

Plant regeneration. Plant tissue treated by particle acceleration areplated on modified MS media supplemented with 13.3 μM benzylaminopurine,0.2 μM naphthalene acetic acid, 5 μM thiamine and 12 mM proline andincubated in the dark for 1-2 weeks, at room temperature. The axes arethen transferred to fresh MS media supplemented with 1.7 μMbenzylaminopurine and 0.2 μM indolyl-3-butyric acid. Plant regenerationis allowed to proceed by continuous incubation of the axes under the 16h photoperiod. Multiple shoots are formed from both the primary andaxillary meristems.

Excised shoots are rooted for further growth by plating them on plantregeneration medium.

Stability, heritability and immunogenicity analyses or GtfB protein areperformed as described in Examples 1 and 2.

While the invention has been disclosed by reference to the details orpreferred embodiments, the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

What is claimed is:
 1. A transgenic plant, comprising and expressing aDNA sequence coding for an antigen of a pathogenic microorganism or anantigenic determinant thereof, said antigen or antigenic determinantthereof eliciting a secretory immune response in a human or other animalupon oral administration of tissue of said plant.
 2. The plant of claim1 wherein the DNA sequence comprises any gene, combination of genes,gene fragment, or combination of gene fragments coding for said antigen.3. The plant of claim 1 wherein the DNA sequence is a synthetic sequencecoding for said antigen.
 4. The plant of claim 1 wherein said antigen isfrom Streptococcus mutans or Escherichia coli.
 5. The plant of claim 1wherein the antigen is selected from the group consisting of SpaA, GtfB,dextranase, K88, K99, CFA, LT-B or an antigenic determinant thereof. 6.The plant of claim 1 which is dicotyledonous.
 7. The plant of claim 1which is monocotyledonous.
 8. A comparison suitable for eliciting asecretory immune response in a human or other animal which comprises:atransgenic plant or tissue obtained from a transgenic plant, saidtransgenic plant comprising and expressing a DNA sequence coding for anantigen of a pathogenic microorganism or an antigenic determinantthereof, said antigen or antigenic determinant being capable ofeliciting a secretory immune response upon oral administration of saidplant or plant tissue.
 9. The composition of claim 8 wherein the DNAsequence comprises any gene, combination of genes, gene fragment, orcombination of gene fragments coding for said antigen.
 10. Thecomposition of claim 8 wherein the DNA sequence is a synthetic sequencecoding for said antigen.
 11. The composition of claim 8 wherein saidantigen is from Streptococcus mutans or Escherichia coli.
 12. Thecomposition of claim 8 wherein the antigen is selected from the groupconsisting of SpaA, GtfB, dextranase, K88, K99, CFA, LT-B or anantigenic determinant thereof.
 13. The composition of claim 8 whereinthe transgenic plant is dicotyledonous.
 14. The composition of claim 8wherein the transgenic plant is monocotyledonous.
 15. The composition ofclaim 8 which further comprises an immunologically acceptable adjuvant.16. A transgenic plant which(a) expresses a DNA sequence coding for anantigen or antigenic determinant of a pathogenic microorganism, and (b)comprises plant tissue which after being orally administered to a humanor other animal induces a secretory immune response to said antigen orantigenic determinant.
 17. A composition comprising tissue of thetransgenic plant of claim 16 wherein said plant or tissue from saidplant is admixed with a food substance.
 18. A composition comprisingtissue of the transgenic plant of claim 1 wherein said plant or tissuefrom said plant is admixed with a food substance.
 19. The transgenicplant of claim 16 wherein said pathogenic microorganism is Streptococcusmutans or Escherichia coli.
 20. Tissue of the plant of claim
 1. 21.Tissue of the plant of claim
 2. 22. Tissue of the plant of claim
 3. 23.Tissue of the plant of claim
 4. 24. Tissue of the plant of claim
 5. 25.Tissue of the plant of claim
 6. 26. Tissue of the plant of claim 6.